Spofihr,  Herman  Augustus 

THE  CARBOHYDRATE  ECONOMY  OF  CACTI 


435 


INnOWVlAVD 


THE  CARBOHYDRATE  ECONOMY 
OF  CACTI 


BY 
HERMAN  AUGUSTUS  SPOEHR 


PUBLISHED  BY  THE  CARNEGIE  INSTITUTION  OF  WASHINGTON 
WASHINGTON,  1919 


CARNEGIE  INSTITUTION  OF  WASHINGTON 
PUBLICATION  No.  287 


«  Jsorfc  (gafttmori  (pr«e« 

BALTIMORE,  IID.,  U.  8.   A. 


en 


PREFACE. 

The  purpose  of  this  work  has  been  primarily  to  gather  data  and  some 
necessary  facts  which  could  be  brought  to  converge  for  an  attack  on  the 
problems  of  photosynthesis.  Experiments  which  have  been  in  progress  in 
the  Desert  Laboratory  at  Tucson  for  several  years  have  indicated  clearly 
that  prerequisite  to  a  rational  discussion  of  the  problems  concerning  the 
manner  in  which  sugars  are  formed  in  the  chlorophyllous  leaf  is  a  clearer 
understanding  of  the  conditions  governing  the  equilibria  and  mutual  trans- 
formations of  the  various  groups  of  carbohydrates  in  the  leaf,  as  well  as  of 
the  fate  of  these  substances  in  the  general  metabolism.  The  complex  and 
manifold  character  of  the  phenomenon  of  photosynthesis  has,  it  would 
seem,  not  been  very  generally  realized  by  workers  in  this  field.  The  avenue 
of  approach  which  appears  most  promising,  at  this  stage  at  least,  is  that 
which  employs  chemical  methods  and  conceptions.  However,  in  following 
this  line  of  attack,  the  dangers  of  reasoning  from  knowledge  gained  purely 
from  in  vitro  investigations  can  hardly  be  overemphasized.  Furthermore, 
consistent  and  valid  results  with  living  material  can  be  gained  only  by  the 
most  careful  control  of  conditions,  such  as  temperature,  water  relations, 
light,  and  the  previous  history  of  the  plant.  While  it  is,  of  course,  highly 
desirable  to  know  the  behavior  of  plants  in  the  field  and  to  develop  methods 
for  gaining  such  knowledge,  it  seems  to  the  writer  that  a  realization  of  this 
ideal  can  be  hoped  for  only  after  the  more  fundamental  principles  of  the 
phenomenon  have  been  learned.  In  the  study  of  photosynthesis  it  is  im- 
possible to  segregate  entirely  the  activity  of  sugar  synthesis  from  that  of 
the  further  metabolic  transformations  and  from  the  catabolic  processes 
which  yield  energy  to  the  living  organism,  glycolysis.  This  latter  phe- 
nomenon has  received  attention  from  many  sides,  and  the  application  of  the 
chemical  point  of  view  to  the  manner  of  sugar  disintegration  and  rearrange- 
ment seems  most  promising  as  an  aid  to  a  clearer  understanding  of  the 
nature  and  mode  of  the  sugar  break-down  in  the  living  cell. 

This  paper  comprises  the  results  of  investigations  carried  out  during 
1916-1918.  The  work  consisted  largely  of  the  analysis  of  plants  which  had 
been  subjected  to  various  experimental  conditions.  Of  the  large  number  of 
analyses  made,  only  those  are  discussed  here  which  are  pertinent  to  the 
immediate  subject.  It  is  a  pleasure  to  acknowledge  here  the  assistance 
rendered  in  this  work  by  Dr.  J.  M.  McGee  and  Mr.  R.  A.  Burt. 

DESERT  LABORATORY, 

Tucson,  Arizona,  December  1918. 


CONTENTS. 

Page. 

I.  Introductory  discussion   5 

II.  Historical    24 

III.  Experimental  methods: 

Selection  and  preparation  of  material 26 

Preparation  of  material  for  estimation  of  sugars 26 

The  determination  of  dry  weight 27 

Preparation  of  material  for  analysis 27 

Calculation  of  analytical  data 30 

The  analysis  of  the  sugar  solutions 31 

The  process  of  reduction 33 

The  determination  of  copper 34 

The  standardization  of  the  solutions 35 

The  estimation  of  the  pentose  sugars 36 

CO,  determination  38 

IV.  The  carbohydrates  of  the  Cacti 39 

V.  Seasonal  variations  in  the  carbohydrate-content 48 

VI.  Effect  of  water  on  the  carbohydrate-content 57 

VII.  Effect  of  temperature  on  the  carbohydrate-content 61 

VIII.  Aerobic  and  anaerobic  respiration 64 

IX.  Consumption  of  carbohydrates  during  starvation 70 

X.  Origin  and  r61e  of  pentose  sugars 75 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


By  HERMAN  AUGUSTUS  SPOEHR. 


I.  INTRODUCTORY  DISCUSSION. 

The  best  evidence  gained  since  the  time  of  Sachs  points  to  the  conclusion 
that  sugars  are  the  first  products  which  accumulate  in  the  process  of  the 
photosynthesis  of  carbon  compounds  in  the  chlorophyllous  cell.  Thus, 
sugars  may  be  considered  the  starting-point  for  the  synthesis  of  the  tre- 
mendous number  of  substances  found  in  living  things,  both  vegetable  and 
animal.  What  the  sugar  or  mixture  of  sugars  is  which  thus  commands  the 
center  of  attention  in  the  metabolism  of  plants  and  of  almost  all  living 
things  is  still  a  question  of  much  dispute  and  uncertainty;  nor  does  the 
solution  of  this  problem  seem  possible  until  we  have  gained  more  knowledge 
of  the  transformations  which  the  various  sugars  undergo  in  the  cell,  inde- 
pendent of  photosynthesis.  It  has  long  been  known  that  in  the  leaf,  under 
circumstances,  the  polysaccharides  are  converted  into  the  simpler  sugars 
and  vice  versa,  by  what  appears  to  be  the  shifting  of  an  equilibrium  by 
means  of  enzyme  action.  It  is  self-evident  that  a  knowledge  of  these  trans- 
formations must  be  obtained  before  the  problem  of  the  first  sugar  syn- 
thesized can  be  attacked. 

Not  only  in  relation  to  the  question  of  the  immediate  products  of  photo- 
synthesis is  the  study  of  the  carbohydrate  equilibrium  important,  but  also 
to  the  question  of  metabolism.  In  a  very  large  number  of  plants,  especially 
the  higher  ones,  both  the  living  and  the  lifeless  matter  consists  in  the  main 
of  material  of  carbohydrate  nature.  The  lifeless  matter  forms  the  walls, 
vessels,  supporting  frame-work,  and  often  a  considerable  amount  of  the 
reserve  food-material.  Those  portions  of  the  plant  in  which  the  manifesta- 
tions of  life  appear,  as  for  instance  the  chromatin  of  the  nucleus  and  the 
protoplasm  itself,  contain  a  considerable  quantity  of  carbohydrates,  the  life- 
less being  formed  from  the  living  and  the  living  drawing  upon  the  lifeless 
for  support.  It  is,  however,  a  question  whether  the  difference  between  the 
living  and  lifeless  is  essentially  one  of  chemical  constitution.  The  idea  that 
the  living  substance  or  protoplasm  is  a  complex  compound  of  more  or  less 
definite  chemical  constitution  no  longer  seems  tenable.  Protoplasm,  a 
mixture  of  so  many  different  substances,  undoubtedly  varies  in  composition 
in  different  organisms.  It  is  in  all  probability  the  study  of  the  physical 
and  chemical  properties  of  protoplasm  as  a  colloidal  mixture  (such  proper- 
ties as  imbibition  of  water,  electrical  charges,  and  surface  phenomena) 
which  will  yield  the  most  illuminating  results. 

5 


6  THE  CARBOHYDRATE  ECONOMY  OF   CACTI. 

It  seems  highly  improbable  that  the  life  processes  consist  of  any  one 
series  of  chemical  changes  or  are  dependent  upon  any  particular  molecule 
or  chemical  group.  But  rather  the  simpler  life  processes  entailing  energy 
changes  may  perhaps  be  regarded  as  a  complex  of  interrelated  chemical 
changes  taking  place  in  a  certain  medium  or  substratum.  This  medium, 
colloidal  dispersion,  mixture,  or  aggregate  of  various  substances  is  the  seat 
or  substratum  in  which  the  various  chemical  reactions  take  place,  the  nature 
and  course  of  which  are  determined  by  the  complex  of  properties  associated 
with  water  control,  surface  phenomena,  and  of  course  catalysts  (such  as 
inorganic  salts  and  enzymes).  These  colloids  do  not  enter  into  or  support 
the  chemical  reactions  or  do  so  probably  only  in  a  rather  indirect  manner, 
serving  primarily  as  a  physical  medium.  Such  a  system  would  be  of  a 
heterogeneous  nature  and,  of  course,  of  the  most  complex  type,  and  capable 
of  various  adsorption  phenomena  productive  of  localized  action  which  would 
influence  the  function  as  well  as  the  structure  of  the  system.  In  fact,  such 
a  hypothesis  would  demand  that  the  substratum  be  relatively  stable,  that  the 
colloidal  material  once  formed  does  not  break  down  as  readily  as  the  other 
substances,  or  only  after  the  supply  of  these  has  been  exhausted.  This  does 
not  mean  actual  or  chemical  stability,  but  rather  relative  to  the  other  sub- 
stances under  the  existing  physiological  conditions,  as,  for  instance,  rela- 
tively slightly  dissociated  by  salts  or  other  catalysts  and  resistant  to  the 
action  of  enzymes. 

This  colloidal  material  may  also  in  part  be  composed  of  substances  which 
do  not  undergo  complete  disintegration  and  thus  are  not  excreted  from  the 
cell.  The  proteins  in  most  plants,  due  to  the  high  synthetic  power  charac- 
teristic of  plants,  are  rarely  so  drastically  affected  that  they  are  not  again 
reconstituted;  these  substances  are  thus  relatively  stable  in  the  sense  that 
under  normal  conditions  they  do  not  disappear  in  the  course  of  metabolism. 
The  cell  substratum  or  protoplasm  should  then  be  regarded  as  a  complex  of 
substances  of  relative  stability  in  which  the  more  sensitive  substances  break 
down  with  the  liberation  of  energy,  the  formation  of  products  of  catabolism, 
and  the  synthesis  of  other  more  complex  substances  in  varying  amounts. 
It  is,  then,  the  combination  of  the  intricate  chemical  reactions  taking  place 
in  this  heterogeneous  colloidal  system  that  constitutes  the  principle  of 
energy  change  of  living  processes.  While  the  chemical  reactions  are  influ- 
enced by  the  nature  of  the  medium,  the  latter  is  also  a  product  of  the 
chemical  processes. 

The  conception  of  protoplasm  as  a  tremendously  complex  "  living  "  pro- 
tein molecule  arose  before  any  considerable  chemical  knowledge  of  this 
group  of  substances  had  been  gained.  Through  the  extensive  researches  of 
Fischer,  Kossel,  Abderhalden,  and  many  others  it  has  become  apparent 
that  the  conception  of  such  an  enormous  complexity  and  sensitiveness  of  the 
proteins  was  rather  unwarranted.  Furthermore,  the  theory  that  proteins, 
carbohydrates,  and  fats  are  synthesized  into  elaborate  complex  substances 
before  they  break  down  and  yield  energy  was  necessitated  by  the  prevalent 


INTRODUCTORY  DISCUSSION.  7 

idea  of  the  relative  stability  of  these  substances  in  simple  solution.  It  is 
now  becoming  more  evident  that  under  conditions  such,  as  exist  in  an 
organism  (i.  e.,  in  the  presence  of  various  catalysts  such  as  inorganic  salts, 
enzyme,  acids,  and  alkalies),  the  molecules  of  proteins,  fats,  and  especially 
carbohydrates  break  apart  almost  spontaneously.  From  a  vast  amount  of 
physiological  evidence  now  at  hand,  it  appears  that  the  energy  of  an 
organism  is  derived  chiefly  from  the  break-down  of  the  material  ingested 
as  food.  When  the  supply  of  nutritive  material  is  insufficient  the  nature 
of  the  respiration  changes  and  then,  in  all  probability,  the  more  stable 
substratum  is  drawn  upon.  This  becomes  evident  in  the  cacti  as  well  as  in 
many  other  plants. 

Kosinski1  has  shown  that  when  Aspergillus  niger  is  grown  on  pure  water, 
the  carbon  dioxid  given  off  falls  rapidly  to  a  value  about  one-quarter  of 
that  when  grown  on  sugar.  During  this  time  the  organism  is  probably 
drawing  on  the  more  stable  protoplasmic  substances.  When  sugar  is  made 
available,  the  carbon  dioxid  rises  immediately,  indicating  the  direct  utiliza- 
tion of  that  substance. 

The  colloidal  materials  found  in  plants  are  in  general  relatively  chemi- 
cally stable  substances,  although  their  exact  chemical  nature  and  mode  of 
formation  as  yet  have  not  been  definitely  established.  It  now  seems  prob- 
able that  in  a  sense  they  represent  by-products  or  end-products  of  the 
metabolic  process.  The  nature  of  this  material  undoubtedly  varies  in 
different  plants,  especially  in  regard  to  the  proportion  of  carbohydrate  and 
proteinaceous  substances.  This  is  indicated  in  the  study  of  the  behavior 
of  the  colloids  of  various  plants.  Extensive  comparative  investigations  with 
different  vegetable  tissues,  prepared  "  biocolloids,"  and  especially  with  the 
cacti,  have  indicated  that  these  plants  behave  like  masses  of  gels  composed 
largely  of  colloidal  carbohydrates.* 

In  the  present  investigation  an  effort  has  been  made  to  determine  the 
nature  of  the  carbohydrates  of  the  cacti  and  to  study  the  transformations 
which  these  undergo  under  various  conditions.  In  the  conception  of  the 
colloidal  nature  of  protoplasm  briefly  outlined,  it  has  been  stated  that  this 
is  of  necessity  a  relatively  stable  system  and  that  the  colloidal  material  is 
itself  a  product  of  metabolism.  One  of  the  most  striking  and  interesting 
features  of  these  plants  is  the  presence  of  large  quantities  of  pentosans  and 
mucilaginous  material.  In  the  cacti  it  has  been  found  that  water  imbibition, 
swelling,  and  growth  are  intimately  related  to  the  presence  of  pentose 
polysaccharides.  The  pentosans  have  been  found  as  components  in  varying 

1  KOSINSKI,    I.      Die   Atmung   bei    Hungerzustanden    und    unter    Einwirkung   von 

mechanischen  und  chemischen  Reitzmitteln  bei  Aspergillus  niger.    Jahrb. 

wiss.  Bot.,  37,  137-204,  1898. 
'MAcDouGAL,  D.  T.,  and  H.  A.  SPOEHB.    The  behavior  of  certain  gels  useful  in  the 

interpretation  of  the  action  of  plants.    Science,  n.  s.,  45,  484-488,  1917. 

, .    Growth  and  imbibition.    Proc.  Amer.  Phil.  Soc.,  44,  289,  1917. 

MAcDouoAL,  D.  T.    Imbibitional  swelling  of  plants  and  colloidal  mixtures.    Science, 

44,  502-505,  1916. 
McGEE,  J.  M.    The  imbibitional  swelling  of  marine  algae.    The  Plant  World,  21, 

13-15,  1918. 


8  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

amounts  of  almost  all  plants.  In  the  cacti  conditions  are  unusually  favor- 
able for  the  study  of  the  formation  and  nature  of  these  substances.  It  is  not 
claimed  that  the  pentosans  in  themselves  represent  the  composition  of 
protoplasm.  The  interest  lies  in  the  transformations  which  the  carbo- 
hydrates undergo  in  the  course  of  metabolism  under  varying  external  con- 
ditions and  the  nature  of  the  chemical  changes  which  are  involved  in  the 
economy.  Nor  are  these  phenomena  to  be  considered  as  a  complete  account, 
but  at  the  most  as  a  single  chapter  in  a  very  extensive  and  involved  story. 

It  would  seem  that  the  material  used  in  respiration  of  most  of  the  higher 
plants  is  essentially  of  carbohydrate  nature.  Whether  plants  use  only 
carbohydrates  in  the  course  of  normal  respiration  has  not  been  established. 
Fats,  of  course,  are  used  extensively  by  plants,  especially  in  seeds;  but 
whether  the  fat  is  converted  into  carbohydrate  before  it  is  used  in  the 
respiratory  process  is  still  unsettled.  As  to  the  role  of  proteins,  especially 
in  the  higher  plants,  we  are  quite  in  the  dark,  but  it  can  be  assumed  with 
safety  that  these  substances  do  not  play  nearly  so  important  a  part  in  the 
economy  of  higher  plants  as  they  do  in  animals.  As  will  be  shown,  of 
greatest  importance  to  the  mode  of  the  breaking  down  of  the  simpler  carbo- 
hydrates is  the  nature  or  condition  of  solution.  In  the  investigations  to  be 
cited  this  condition  refers  more  especially  to  the  degree  of  alkalinity.  There 
is  considerable  reason  for  believing  that  the  proteins  and  their  simpler 
derivatives,  incorporated  in  this  cell  substratum  or  medium,  produce  therein 
the  necessary  conditions  under  which  glycolysis  may  proceed,  or  through  the 
production  of  acid  and  alkaline  products  regulate  the  enzymatic  activities. 
These  products  are  in  turn  again  synthesized  into  the  protoplasmic  proteins, 
so  that  relatively  small  amounts  of  protein  suffice  for  the  conversion  of  large 
quantities  of  carbohydrate  and  (as  actually  seems  to  be  the  case)  the  major 
portion  of  the  energy  released  is  thus  to  be  ascribed  to  the  catabolism  of  the 
carbohydrates.  Also,  the  remarkable  property  possessed  by  the  proteins 
and  their  salts  of  neutralizing  acids  or  bases  by  the  opening  up  of  suc- 
cessive N.H-O-C  groups  gives  them  the  power  of  maintaining  the  H*  or 
OH"ion  concentration  of  the  medium  within  definite  limits,  usually  close 
to  neutral.  In  view  of  the  fact  that  the  products  of  carbohydrate  catabo- 
lism are  mostly  acid,  it  might  well  be  expected  that  this  property  is  of  prime 
physiological  importance,  and  that  herein  lie  some  of  the  fundamental 
differences  between  cells  of  animals  and  those  of  plants  with  their  vacuolar 
fluid.  Some  workers,  in  fact,  have  ascribed  to  the  proteins  themselves 
enzymatic  properties  which  may  be  interpreted  that  the  zymogen  as  well  as 
the  co-enzyme  is  a  product  of  the  protein.  These  conceptions  evidently 
depend  upon  a  clearer  understanding  of  the  nature  of  enzyme  action  and 
catalysis  before  any  substantiation  or  further  elucidation  may  be  hoped  for. 

Deleano,1  working  with  mature  leaves  of  Vitis  vinifera  at  18  to  22° 
in  the  dark,  found  that  these  utilize  only  carbohydrates  during  the  first 

1  DELEANO,   NICOLAS   T.    Studien   ueber   den   Atmungsstoffwechsel   abgeschnittener 

Laubblatter.    Jahr.  f.  wiss.  Bot.,  51,  541-592,  1912. 

BOBODIN,  J.    Ueber  die  physiologische  Rolle  und  die  Verbreitung  des  Asparagins  im 
Pflanzenreiche.    Bot.  Zeitg.,  36,  801-832,  1878. 


INTRODUCTORY  DISCUSSION.  9 

100  hours.  During  this  time  the  amount  of  protein  and  other  nitrogen 
compounds  in  the  leaves  does  not  change;  thereafter,  and  when  all  carbo- 
hydrate has  disappeared,  the  nature  of  the  respiratory  process  changes 
radically:  the  proteins  are  broken  down,  a  variety  of  non-protein  nitro- 
genous substances  appears,  and  ammonium  salts  are  formed.  It  is  a  matter 
of  common  knowledge  that  when  a  fungus  is  fed  solely  on  proteinaceous 
material  there  is  formed  a  large  amount  of  ammonia,  and  that  this  is  com- 
pletely reduced  or  inhibited  when  sugar  is  made  available  to  the  organism. 
These  phenomena  indicate  the  widely  different  paths  which  the  course  of 
metabolism  may  follow  under  varying  conditions  and  which,  unfortunately 
for  a  clearer  understanding  of  the  subject,  have  been  ascribed  to  obscure 
regulatory  devices  of  the  organism.  They  furthermore  suggest  the  exist- 
ence of  a  condition  in  the  nature  of  a  balance  between  or  interdependence  of 
carbohydrate  and  protein  catabolism  in  the  mature  vegetable  organism.  In 
young  and  growing  portions  of  a  plant  the  state  of  affairs  is  of  course  quite 
different  and  more  complex. 

In  spite  of  the  enormous  amount  of  work  which  has  been  done  and  its 
great  economic  importance,  no  definite  conclusion  has  been  reached  even 
for  the  mammalian  organism.  Thus,  to  quote  Lusk  (Science  of  Nutrition, 
p.  277,  1917) : 

"  Thomas  calculated  that  during  the  period  of  minimal '  wear-and-tear '  pro- 
tein metabolism,  0.4  calories  were  derived  from  the  metabolism  of  1.5  milligrams 
of  protein  per  kilogram  of  body-weight  every  hour,  while  0.96  calories  were 
derived  from  the  oxidation  of  259  milligrams  of  glucose.  In  other  words,  pro- 
tein furnished  only  4  per  cent  of  the  energy  required  by  a  man  at  rest.  Since 
mechanical  work  scarcely  influences  the  *  wear-and-tear '  quota  of  protein 
metabolism,  although  it  largely  increases  the  oxidation  of  carbohydrate,  it  is 
evident  that  protein  may  play  a  very  small  role  as  a  producer  of  energy  for  the 
maintenance  of  the  function  of  life.  When  carbohydrates  are  given  in  the  diet, 
it  is  possible  to  establish  nitrogen  equilibrium  at  a  much  lower  level  than  when 
protein  alone  or  protein  and  fat  are  ingested." 

Besides  furnishing  methods  for  the  study  of  metabolic  products,  chem- 
istry has  been  able  to  be  of  service  to  physiology  largely  because  it  has  sup- 
plied many  valuable  analogies  between  chemical  phenomena  outside  the 
cell  and  the  processes  in  the  living  organism  itself.  These  analogies  are  of 
closer  relation  than  mere  resemblance  or  likeness,  and  can  with  profit  be 
reasoned  from,  so  that  it  is  safe  to  infer  that  other  and  often  deeper  rela- 
tions exist.  Such  analogies  have  aided  us  greatly  in  our  thinking  and  have 
influenced  strongly  the  formulation  of  the  theories  of  many  vital  processes. 
The  method  is,  of  course,  open  to  some  criticism,  and  its  value  depends  upon 
the  real  identity  in  important  aspects  of  the  cases  compared.  Physiological 
experimentation  is  often  associated  with  such  great  difficulties  that  its 
course  can  be  opened  only  after  the  simpler,  purely  chemical  relationships 
have  been  formulated.  Then,  as  has  often  been  the  case,  physiological 
investigation  contributes  to  a  clearer  understanding  of  the  chemical  phe- 
nomena and  the  thread  which  binds  the  two  is  found  to  be  the  identity  of 
a  general  principle. 


10  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

For  the  problems  of  carbohydrate  metabolism,  and  especially  for  glycoly- 
sis,  some  recent  investigations  on  the  dissociation  and  molecular  rearrange- 
ments of  the  sugars  under  various  conditions  have  been  most  suggestive. 
No  application  of  these  newer  conceptions  has  been  made  in  the  interpreta- 
tion of  the  metabolic  processes  in  the  plant  The  more  important  principles, 
therefore,  are  given  here,  as  it  seems  to  the  writer  that  these  ideas  are  among 
the  most  valuable  which  chemistry  has  given  to  physiology.  For  the  more 
detailed  exposition  and  proof,  reference  must  be  made  naturally  to  the 
rather  voluminous  original  works.  A  thorough  knowledge  of  the  chemistry 
of  the  substances  concerned  in  these  processes  is,  of  course,  essential  to  an 
understanding  of  the  physiology.  The  attention  can  be  confined  to  the 
reactions  in  aqueous  solution,  as  it  is  in  this  state  that  the  many  trans- 
formations occur  in  the  living  organism.  Pure  solutions  of  the  mono- 
saccharides  in  vitro  are  comparatively  stable.  In  living  matter  they  are 
very  unstable  and  capable  of  the  most  multifarious  rearrangements  and 
disintegrations.  Some  insight  can  be  gained  into  the  causes  of  this  insta- 
bility of  sugars  in  living  matter  from  a  consideration  of  the  manner  in 
which  these  substances  dissociate  under  the  influence  of  various  reagents 
and  the  products  which  result  from  such  reactions. 

Organic  compounds  containing  one  or  more  hydroxyl  groups,  such  as 
alcohol,  glycerine,  or  glucose,  exhibit  some  of  the  properties  of  exceedingly 
weak  acids.  They  form  salts  with  metals  and  influence  the  electrical  con- 
ductivity of  weak  electrolytes  as  other  weak  acids  do.  The  electrolytic 
dissociation  constants  of  several  of  these  substances  have  been  measured  by 
electro-chemical  methods.  Thus  Michaelis  and  Eona1  give  the  following 
constants  at  18°: 


Ethyl  alcohol <  10-" 

Glycerine    =      7  X  10-» 

Mannlt    =    3.4  X  10-" 

Dulcit    =    3.5  X  10-" 

Galactose    =    5.2  X  10-" 

Fructose   =    9.0  X  10-" 


Mannose    =  1.09  X  10-" 

Glucose    =    6.6  X  10-" 

Maltose    =18.0X10-" 

Saccharose   =   2.4  X  10-" 

Lactose    =    6.0  X  10-" 

Raffinose     =    1.8  X  10-" 


That  sugars  are  very  weak  acids  can  be  seen  by  comparison  with  the 
dissociation  constants  of  the  following  common  organic  acids : 


K  for  18e 


Acetic  acid 1.8  X  10-» 

Butyric  acid 1.5  X  10-' 

Lactic  acid  ..  ,..1.4  X  1Q-4 


Carbonic  acid  (H:HC(V) . .  .3.04  X  10~T 

Tartaric  acid 1  X  10-» 

Phenol    .  . .  5.8  X  10-" 


Salts  in  which  a  metal  replaces  the  hydrogen  in  the  hydroxyl  group  of 
the  alcohols  and  sugars  have  been  known  for  some  time.  Thus  sodium 
ethylate  (C2H5ONa)  can  be  readily  obtained  by  the  action  of  metallic 
sodium  on  absolute  alcohol.  Glycerine  also  forms  similar  compounds,  the 
glycerates,  with  a  large  number  of  metals.  Here  there  are  three  replaceable 

1  MICHAELIS,  L.,  and  P.  RONA.  Die  Dissociationskonstanten  einiger  sehr  schwacher 
saeuren,  insbesondere  der  Kohlenhydrate  gemessen  auf  elecktrometrischem 
wege.  Biochem.  Zeit.,  49,  232-248,  1913. 


INTRODUCTORY  DISCUSSION.  11 

hydrogen  atoms.  In  the  same  group  fall  the  metallic  derivatives  of  glucose 
and  other  sugars;  these  are  already  decomposed  by  such  weak  acids  aa 
carbonic  acid.  For  the  present  purpose  the  most  important  property  of 
these  metallic  compounds  is  the  fact  that  they  decompose  very  readily  ;  far 
more  easily  than  the  original  alcohol  or  sugar.  Thus,  Nef  *  has  shown  that 


T> 

the  salts  of  primary  and  secondary  alcohols  -R>CHOM  (M=K,  N"a,  Ca, 

Ba,  or  Zn)  dissociate  at  180°  to  250°,  while  the  corresponding  free  alcohols 
dissociate  at  much  higher  temperatures,  400°  to  600°.  Hence  the  salts  are 
much  less  stable  and  burn  spontaneously  in  the  air,  while  the  free  alcohols 
are  quite  stable. 

A  great  deal  of  work  has  been  done  on  the  effect  of  alkalies  and  various 
salts  on  the  sugars.*  One  incentive  to  these  investigations  has  been  that  the 
reactions  thus  produced  simulate  in  some  regard  the  action  of  various 
enzymes  on  sugars.  The  principle  involved  in  these  sugar  reactions  is  the 
same  or  very  closely  related  to  the  action  of  the  alcohols  just  indicated.  A 
few  examples  will  suffice  to  illustrate.  An  aqueous  solution  of  glucose  is 
affected  but  very  slowly  by  the  oxygen  of  the  air  or  by  hydrogen  peroxide. 
If,  however,  a  trace  of  a  salt  of  iron,  for  instance,  is  added  to  the  mixture, 
the  glucose  is  oxidized  rapidly  with  the  evolution  of  much  heat  and  the 
formation  of  a  large  variety  of  compounds.  An  exceedingly  small  amount 
of  iron  suffices  to  oxidize  large  quantities  of  sugar. 

In  the  presence  of  alkalies  all  the  monosaccharides  are  unstable  and 
spontaneously  decompose  into  a  large  variety  of  substances.*  If  the  con- 
centration of  the  alkali  employed  is  very  low,  there  takes  place  a  series  of 

*NEF,  J.  U.     Dissociationsvorgaenge  bei  den  einatomigen  Alcoholen,  Aethern,  und 

Salzen.    Liebig's  Annalen  der  Chem.,  318,  138,  1901. 
3  FENTON,  H.  J.  H.    The  oxidation  of  tartaric  acid  in  the  presence  of  iron  salts.  Jour. 

Chem.  Soc.,  65,  899,  1894. 

-  ,  and  H.  JACKSON.    Oxidation  of  polyhydric  alcohols  in  the  presence  of  iron. 

Jour.  Chem.  Soc.,  75,  1-11,  1899. 

-  ,  -  .    The  oxidation  of  organic  acids  in  the  presence  of  ferrous  iron. 

Jour.  Chem.  Soc.,  77,  69-76,  1900. 

-  ,  -  .     Degradation  of  glycolic  aldehyde.    Ibid.,  77,  1294-1298,  1900. 
MOBBELL,  R.  S.,  and  F.  M.  CROFTS.    Action  of  hydrogen  peroxide  on  carbohydrates  in 

the  presence  of  ferrous  salts.  Jour.  Chem.  Soc.,  75,  786-792,  1899;  Ibid.,  77, 
1219-1221,  1900;  Ibid.,  81,  666-675,  1902;  Ibid.,  83,  1284-1292,  1903;  Ibid., 
87,  280-293,  1905. 

RUFF,  OTTO.  Ueber  die  Verwandlung  der  D-glucose  in  D-Arabinose.  Ber.  chem. 
Ges.,  31,  1573-1577,  1898. 

-  .     D-  und  R-Arabinose.    Ibid.,  32,  550-560,  1899. 

-  .     D-Erythrose.     Ibid.,  32,  3672-3681,  1899. 

OLLENDOBF,  D.  G.  Abbau  von  D-Galactose  und  von  Michzucker  (D-  und  Galactoara- 
binose).  Ibid.,  33,  1798-1810,  1900. 

-  .     Ueber   die   Oxidation  der   1-Arabensaeure  und  1-xylonsaeure.     Ibid.,   34, 

1362-1372,  1901. 

-  .    Ueber  den  Abbau  der  Rhamnon  und  Isosaccharin  Saeure.    Ibid.,  35,  2360- 

2370,  1902. 

SPOEHB,  H.  A.  On  the  behavior  of  the  ordinary  hexoses  towards  hydrogen  peroxide 
in  the  presence  of  alkaline  hydroxides,  as  well  as  of  various  iron  salts. 
Amer.  Chem.  Jour.,  43,  248-254,  1910. 

*NEF,  J.  U.     Dissociationvorgaenge  in  der  Zuckergruppe:   III.     Liebig's  Annalen  der 
Chem.,  403,  204,  1913. 


12 


THE   CARBOHYDRATE  ECONOMY  OF   CACTI. 


complex  reciprocal  transformations  of  the  hexoses  as  discovered  by  Lobry 
de  Bmyn  and  van  Ekenstein.1  Here  the  sugar  is  not  decomposed,  but  under 
the  influence  of  the  alkali  the  structural  arrangement  of  the  sugar  molecule 
is  affected  in  such  a  manner  as  to  produce  a  number  of  substances  which 
differ  from  each  other  only  in  respect  to  the  position  of  certain  chemical 
groups  within  the  molecule.  The  nature  of  these  most  important  reactions 
has  been  worked  out  by  Nef.  He  demonstrated  that  an  aqueous  solution 
of  either  d-glucose,  d-mannose,  or  d-levulose  with  1/20  equivalent  of  calcium 
hydroxide  attains  equilibrium  when  kept  at  15°  to  20°.  There  is  thus 
formed,  when  starting  with  any  one  of  the  above  sugars,  a  mixture  of — 


OH 


OH       OH 


CH2OH 


CH:0,          CH2OH- 


OH       OH 


OH 


OH       OH 


-CH:0 


D-glucose. 
H          H        OH        O 


CHaOH- 


OH       OH 


-CH,OH,        CHaOH 


I 

D- 
I          I 

manno 

i       : 

se. 
I         ( 

) 

CH2OH 


OH       OH       OH 


D-fructose. 

H         O         OH 


CH,OH- 


OH       OH 


-CH..OH,        CH,OH 


p 

D-pse 
[         I 

ado  f  n 
I         ( 

ictose. 
)          E 

CH2OH 


OH       OH 


OH 


and  a-  and  /?-  d-glutose. 

In  a  similar  manner  the  d-galactose  series  yields  d-galactose,  d-talose, 
d-tagatose,  d-sorbose,  and  a-  and  ft-  d-galtose.  It  is  noteworthy  that  a  mem- 
ber of  the  d-glucose  series  is  never  converted  into  a  member  of  the  d-galactose 

1  LOBBY  DE  BBUTN,  C.  A.,  and  W.  A.  VAN  EKENSTEIN.  Action  des  alcalis  sur  les  sucres. 
II:  Transformation  r6ciproque  des  uns  dans  les  autres  des  sucres  glucose, 
fructose  et  mannose.  Rec.  des  trav.  chim.  des  Pays-Bas.,  14,  203-216,  1895. 

,  .     Action  des  alcalis  sur  les  sucres.    Ill:   Transformations  des  sucres 

sous  1'influence  de  1'hydroxide  de  plomb.    Rec.  des  trav.  chim.  des  Pays-Bas., 
15,  92-96,  1896. 

Action  des  alcalis  sur  les  sucres.    IV:  Remarques  generates.    Rec. 
des  trav.  chim.  des  Pays-Bas.,  16,  257-273,  1897. 


INTRODUCTORY  DISCUSSION.  13 

series.  The  difference  in  structure  of  these  two  sugars  lies  especially  in  the 
space  relation  of  the  hydrogen  and  hydroxyl  about  the  fourth  and  fifth 
carbon  atom  in  the  chain  from  the  carbonyl  group.  It  appears,  therefore, 
that  these  reciprocal  transformations  involve  only  the  first  three  carbon 
atoms  of  the  chain,  and  that  there  exists  a  gradient  of  reaction  in  the  sugar 
molecule,  the  highest  being  at  the  carbon  atom  adjacent  to  the  carbonyl 
group  and  decreasing  with  each  carbon  atom  removed  therefrom.  It  is 
interesting  to  compare  the  proportion  of  the  various  sugars  found  in  these 
mixtures  with  the  conditions  as  they  exist  in  nature.  The  majority  of  the 
32  theoretically  possible  aldo-,  2-keto-,  and  3-keto-hexoses  are  now  known 
as  synthetic  products ;  it  is,  however,  a  striking  fact  (which  has  puzzled 
chemists  for  a  long  time)  that  but  a  very  small  number  of  all  these  sub- 
stances is  found  in  plants.  D-glucose  and  d-fructose  are  by  far  the  most 
common  hexoses  and  are  found  in  greatest  abundance,  and  the  only  other 
hexoses  at  all  common  in  plants  are  d-mannose,  d-galactose,  and  1-sorbose. 
In  these  equilibrium  sugar  mixtures,  Nef  found  aldoses  and  ketoses  in 
about  equal  amounts,  but  in  the  glucose  series,  of  the  aldoses  there  were 
present  only  d-glucose  and  d-mannose  and  in  the  proportion  of  5  parts  of 
the  former  to  1  of  the  latter.  In  the  galactose  series  over  90  per  cent  of  the 
aldoses  was  d-galactose.  Furthermore,  no  trace  of  d-allose  nor  d-latose 
on  the  one  hand,  nor  of  1-gulose  or  1-idose  on  the  other  was  ever  formed.  It 
is  most  suggestive  that  the  composition  and  proportion  of  the  various  sugars 
in  equilibrium  found  in  these  experiments  should  so  closely  approach  the 
conditions  existing  in  nature.  For  the  detailed  chemical  dynamics  under- 
lying these  phenomena,  reference  must  be  made  to  the  original  elaborate 
discussion  of  RTef.  However,  the  physiologist  has  herein  for  the  first  time 
the  basis  of  a  rational  explanation  of  this  most  perplexing  problem. 

In  the  presence  of  higher  concentrations  of  alkali  the  sugar  molecule  is 
affected  in  a  more  profound  manner,  and  these  reactions  are  of  special 
interest  in  relation  to  glycolysis  and  carbohydrate  metabolism  in  general. 
It  should  be  stated  that  so  far  the  study  of  plant  juices  and  of  body  fluids 
by  physico-chemical  methods  has  not  revealed  concentrations  of  OH  ions 
such  as  would  be  necessary  to  bring  about  reactions  in  simple  solution  like 
those  under  discussion  here.  At  the  same  time  our  knowledge  of  the  con- 
dition of  plant  protoplasm  as  to  acidity  or  alkalinity  must  be  considered  as 
most  unsatisfactory,  such  determinations  as  have  been  attempted  being  of  a 
very  crude  nature.  It  is  not  implied  that  these  sugar  dissociations  are 
accomplished  necessarily  by  alkaline  hydroxides  or  OH  ions  in  the  organism, 

NOTE. — Nef's  investigations  on  the  composition  of  formose  are  also  of  interest  in 
this  connection.  The  formation  of  formose  (the  mixture  of  sugars  formed 
by  the  action  of  alkalies  on  formaldehyde)  can  be  regarded  as  the  keystone  of 
the  formaldehyde  hypothesis  of  photosynthesis  as  first  suggested  by  Baeyer. 
Nef  found  that  formose  consists  of  a  mixture  half  pentose  and  half  hexose, 
and  from  the  experiments  of  the  action  of  weak  alkalies  on  sugars  he  con- 
cluded that  the  hexoses  in  formose  consist  of  24  members,  i.  e.,  8  aldohexoses, 
and  8  2-ketohexoses  and  8  3-ketohexoses,  and  that  the  aldoses  are  primarily 
dl-glucose  and  dl-galactose. 


14  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

but  rather  by  those  substances  (or  catalysts)  which  under  physico-chemical 
conditions  existent  in  the  cell  exert  on  sugars  an  effect  analogous  or  equiva- 
lent to  that  of  metallic  salts  or  hydroxides  in  aqueous  solution.  In  fact,  the 
importance  and  role  of  catalyzers  in  glycolysis  has  been  established  by  the 
large  amount  of  work  which  has  been  done  with  various  tissue  and  pancreas 
extracts.  Thus,  for  example,  Walter  Loeb1  has  shown  that  substances 
obtained  from  the  alcoholic  pancreas  extracts  by  treatment  with  iron  salts 
effect  a  cleavage  of  glucose  in  which  the  products  formed  were  formaldehyde 
and  pentose.  However,  by  the  use  of  pure  sugar  solutions  and  alkalies,  it 
has  been  possible  to  follow  more  clearly  the  many  complex  disintegrations 
and  rearrangements  which  the  sugar  molecule  undergoes.  The  principle 
of  chemical  action  is  most  probably  the  same.  The  nature  of  the  products 
formed  under  these  conditions  depends  upon  whether  or  not  there  is  present 
an  oxidizing  agent,  such  as  air.  In  the  organism  the  same  differentiation 
exists  in  regard  to  the  catabolism  of  hexoses.  Under  anaerobic  conditions 
one  set  of  products  is  formed,  while  under  aerobic  conditions  there  is 
another  set  of  products  different  from  the  first  results.  The  similarity  of 
the  reactions  obtained  in  vitro  to  those  observed  in  the  organism  of  anaero- 
biosis  and  aerobiosis  are  worthy  of  notice.  In  the  succulent  plants,  such  as 
the  cacti,  conditions  exist  which  lie  between  these  extremes,  i.  e.,  a  low  or 
insufficient  oxygen  supply.1  This  is  perhaps  the  most  important  single 
factor  in  the  formation  of  acid  in  the  plants.  Under  similar  conditions 
there  is  formed  in  the  plant,  not  d-lactic  acid,  but  d-malic  acid.  The  genesis 
of  this  latter  substance  in  succulent  plants  has  been  a  matter  of  much 
speculation,  which,  however,  has  in  no  case  been  founded  on  chemical 
experience.  The  relation  between  anaerobiosis  and  aerobiosis  will  become 
evident  from  the  chemical  discussion  which  is  to  follow. 

If  a  solution  of  d-glucose  is  treated  with  alkali  of  higher  concentration 
than  that  causing  the  reciprocal  transformations,  as  e.  g.,  8  X  normal  sodium 
hydroxide  in  the  absence  of  oxygen  or  an  oxidizing  agent  there  are  formed : 
large  quantities  of  dl-lactic  acid  and  dl  1-3  dioxybutyric  acid,  besides  four 
isomeric  C8  saccharinic  acids.  The  importance  of  lactic  acid  in  the 
metabolism  of  the  mammalian  organism  and  its  formation  under  conditions 
of  insufficient  oxidation  or  restricted  oxygen  supply  is  well  known.*  As 
will  be  shown  later,  the  very  large  quantities  of  pentoses  found  in  these 
plants  arise  from  the  hexoses  under  conditions  of  repressed  metabolic 
activity;  the  further  disintegration  of  the  pentose  sugars  is  therefore  of 
special  interest  in  relation  to  the  present  problem.  That  these  plant  acids 
are  of  great  importance  in  many  of  the  functions  of  the  plant  has  been 
clearly  established  by  Eichards  in  regard  to  the  gaseous  exchange  and 

1  VON  FUEBTH,  OTTO,  and  A.  J.  SMITH.    The  problems  of  physiological  and  pathological 

chemistry  of  metabolism.    Chapt.  XIX,  1916. 
'  RICHARDS,  H.  M.     Acidity  and  gas  interchange  in  cacti.    Carnegie  Inst.  Wash.  Pub. 

No.  209,  p.  32, 1915. 
•  DAKIN,  H.  D.    Oxidations  and  reductions  in  the  animal  body.    Pages  56-68,  85-87, 

1912. 
VON  FUEBTH-SMITH.    L.  c.,  p.  452. 


INTRODUCTORY  DISCUSSION. 


15 


recently  has  also  been  studied  in  relation  to  the  absorption  of  water  and 
growth. 

From  a  study  of  the  action  of  alkalies  on  the  pentoses  in  the  absence  of 
oxygen  or  an  oxidizing  agent,  some  insight  can  be  gained  into  the  formation 
of  malic  acid  in  the  succulents.  When  a  solution  of  1-arabinose  is  thus 
treated  with  eight  times  normal  sodium  hydroxide  there  is  formed  a  variety 
of  acid  products.  Special  interest  is  attached  for  present  consideration  to  the 
dl-1,  3-dioxybutyric  acid:  dl-2,  3-dioxybutyric  acid: 

OH       H  H       OH 


COOH- 


-CH3OH, 


COOH- 


-CH,OH 


By  oxidation  these  acids  go  over  easily  into  d-1  malic  acid.1 

Finally,  to  turn  to  the  action  of  alkalies  or  metallic  salts  on  sugars  in  the 
presence  of  oxygen  or  oxidizing  agents :  The  relative  stability  of  the  sugars 
in  aqueous  solution  toward  oxidizing  agents  and  the  influence  of  small 
quantities  of  metallic  salts  has  already  been  mentioned.  Before  entering 
upon  the  discussion  of  the  way  in  which  alkalies  and  salts  affect  the  sugar 
molecule,  it  will  be  helpful  to  indicate  the  products  which  are  formed  in  the 
oxidation  of  sugars.  The  nature  of  the  products  and  the  relative  propor- 
tion of  these  varies  tremendously  with  different  conditions  and  depends 
upon  a  number  of  factors,  more  especially  the  concentration  of  the  alkali  or 
salt,  the  oxidation  potential,  i.  e.,  whether  the  oxidizing  agent  is  air,  hydro- 
gen peroxide,  cupric  hydroxide,  silver  oxide,  etc.,  and  the  temperature.  The 
different  sugars  also  show  wide  variation  in  the  proportion  of  the  products 
formed.  Thus,  for  example,  certain  sugars  yielded  as  shown  in  table  1 :  * 

TABLE  1. 


C02. 

Formic  acid. 

Oxalic  acid. 

Hydrogen  peroxide  with 
ferric  sulphate: 
D-galactose  
D—  glucose 

p.  ct. 
69.28 
70.98 

p.  ct. 
3.00 
3.00 

p.ct. 
26.90 
16.66 

D-f  ructose  
Hydrogen  peroxide  with 
0.5  normal  KOH: 
D—  galactose  

72.69 
4.65 

0.77 

80.2 

18.89 
0.0 

D-glucose  

3.51 

65.3 

D-f  ructose  

3.58 

48.3 

Air  with  normal  sodium 
hydroxide:8 
D—  galactose  

1.74 

16.70 

Trace 

D  glucose  

14.80 

1  NEF,  J.  U.    Liebig's  Annalen,  376,  13-52,  1910. 
1  SPOEHB,  H.  A.     L.  c.,  p.  234. 
8  NEF,  J.  U.     Liebig's  Annalen,  403,  244,  1913;  Ibid.,  357,  220,  1907;  Ibid.,  357, 
1907. 


16 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


An  alkaline  solution  of  cupric  hydroxide  (Fehling's  solution)  yielded 
from  100  grams  of  the  following  sugars : 


TABLE  2. 


C02. 

HCOOH. 

grams. 

grams. 

D-glucose  

3.36 

12.92 

D-f  ructose  

2.00 

11.40 

D-mannose  

4.40 

12.00 

D-galactose    
D-arabinose   .... 

2.19 
3.40 

13.30 
14.66 

An  alkaline  solution  of  silver  oxide  oxidizes  the  following  sugars  with 
the  formation  of  only  three  products,  carbonic,  formic,  and  oxalic  acids. 
The  oxidation  is  apparently  much  more  drastic  than  with  the  other  oxidiz- 
ing agents;  the  relative  amounts  of  the  three  products  vary  with  the  nature 
of  the  sugar  and  the  concentration  and  temperature  of  the  mixture : 


TABLE  3. 


C02. 

HCOOH. 

(COOH),. 

D—  glucose  

grami. 
22.56 

grams. 

38.45 

grant. 
85.34 

D-f  ructose  
D-galactose  
D-arabinose  

26.00 
29.95 
29.34 

35.30 
43.63 
37.29 

82.70 
64.47 
80.20 

D-glucose  in  alkaline  solution  when  oxidized  with  air  or  with  3'  per  cent 
hydrogen  peroxide  yields  besides  carbonic  and  formic  acids  a  large  amount 
of  non-volatile  acids.  The  latter  are  a  mixture  composed  of  d-arabonic, 
d-erythronic,  1-threonic,  and  dl-glyceric  acids.  The  total  amounts  of  these 
non-volatile  acids,  as  well  as  the  relative  amounts  of  the  individual  acids, 
vary  greatly,  according  to  whether  air  or  hydrogen  peroxide  is  used  as  an 
oxidizing  agent.  Thus  Glattfeld*  obtained  for  every  100  grams  of  glucose 
oxidized  with  H2O2,  23.7  grams  of  the  mixed  non-volatile  acids,  while  with 
air  72  grams  of  the  acid  mixture  were  obtained. 

These  illustrations  have  been  cited  in  order  to  show  the  variability  of  the 
process  of  glycolysis  even  in  vitro.  Among  the  important  variable  factors 
which  influence  enormously  the  course  of  glycolysis  and  the  nature  of  the 
products  are  the  rate  of  oxidation,  concentration,  the  oxidation  potential, 
temperature,  and  undoubtedly  other  less  definite  elements.  It  is  therefore 
not  in  the  least  surprising  that  the  living  organism  in  which  many  of  these 
factors  are  so  closely  interrelated  and  almost  constantly  changing  should 

1  GLATTFELD,  J.  W.  E.    On  the  oxidation  of  d-glucose  in  alkaline  solution  by  air  as 
well  as  by  hydrogen  peroxide.    Amer.  Chem.  Jour.,  50,  135-157,  1913. 


INTRODUCTORY  DISCUSSION.  17 

display  such  a  variety  of  metabolic  products  and  reactions.  As  a  possible 
explanation  of  this  great  instability  and  variability  of  reaction  of  the 
sugars,  the  theories  and  results  of  some  of  the  recent  chemical  investigations 
give  the  most  helpful  suggestions  yet  offered.  It  has  already  been  shown 
that  sugars  behave  like  very  weak  acids,  that  the  hydrogen  atoms  of  the 
hydroxyl  groups  can  be  replaced  by  metals  to  form  salts.  A.  P.  Mathews1 
has  shown  that  the  salts  formed  with  sugars  are  easily  ionized  and  thus  lead 
to  increased  concentration  of  sugar  anions.  These,  by  virtue  of  partially 
unbalanced  charges,  are  very  unstable  and  highly  reactive.  The  most 
reactive  part  of  the  sugar  molecule  is  probably  the  first  carbon  atom,  the 
carbonyl  group ;  thereafter  the  reactivity  decreases  down  the  chain  of  carbon 
atoms,  as  these  are  removed  from  the  carbonyl  group.  While  the  sugars 
are  relatively  stable  substances,  their  salts  decompose  very  readily. 

The  manner  in  which  these  salts  decompose  is  of  greatest  importance  to 
an  understanding  of  glycolysis.  In  this  decomposition  or  dissociation  the 
sugar  molecule  is  broken  in  fragments  composed  of  carbon  atoms  in  varying 
number.  Thus  a  hexose  may  break  into  fragments  containing  5  and  1 
carbon  atoms,  into  fragments  of  2  and  4,  and  into  2  pieces  each  containing 
3  carbon  atoms.  The  proportion  in  which  any  of  these  pieces  are  formed 
depends  upon  a  number  of  factors,  such  as  concentration  of  the  sugar, 
temperature,  etc.  This  dissociation  or  cleavage  of  the  sugars  results  in 
mixtures  of  tremendous  complexity.  Of  special  importance  is  the  fact  that 
these  pieces  are  unstable  and  of  high  reactivity,  so  that  they  react  in  a 
variety  of  ways;  either  the  pieces  undergo  intramolecular  rearrangements 
to  form  more  stable  compounds,  or  they  may  react  with  each  other,  oxidizing 
one  and  reducing  the  other ;  they  may  condense  or  polymerize,  or  finally 
unite  with  other  substances  present,  such  as  oxygen,  to  form  acids.  It  can 
readily  be  seen  what  an  appalling  complex  of  substances  and  tangle  of 
reactions  are  involved  in  a  system  containing,  for  instance,  dextrose  and 
sodium  hydroxide.  Nef  has  found  that  in  such  a  system  there  are  finally 
in  equilibrium  no  less  than  93  different  substances.  Including  the  syntheses 
which  always  accompany  these  reactions,  the  number  of  products  is  con- 
siderably above  100. 

It  is  undoubtedly  due  to  the  ease  and  multifarious  ways  in  which  the 
sugars  are  dissociated  and  undergo  chemical  changes  involving  the  libera- 
tion of  energy  that  they  can  be  used  by  the  organism  both  under  aerobic 
and  anaerobic  conditions.  In  this  respect  neither  the  fats  nor  the  proteins 
can  be  compared  with  the  sugars  as  to  the  possibilities  of  usefulness  for  the 
organism.  It  is  impossible  to  give  here  the  detailed  chemical  steps  used  in 
these  considerations ;  reference  must  be  made  to  the  very  extensive  original 
literature.  The  arguments  are  based  upon  the  following  principles  which 
are  applicable  alike  to  hexose,  pentose,  or  tetrose  sugars :  There  are  formed 

1  MATHEWS,  A.  P.    The  spontaneous  oxidation  of  sugars.    Jour.  Biol.  Chem.,  6,  3, 

1909. 


18 


THE   CARBOHYDKATE  ECONOMY  OF   CACTI. 


(first)  a  series  of  compounds,  the  enols,  in  the  following  manner  (M  in  the 
formulae  represents  a  metal  or  possibly  enzyme),  which  replaces  the  ionized 
hydrogen : 


H     H    OH    H 


H      H     OH     H    H 


CH,OH 


-CH :  0+MOffZ±CHaOH- 


OH   OH    H    OH 

D-glucoae. 
H     H     OH    H     H 


-C— OH- 


OH  OH    H     OH 


H      H     OH 


1 


CHaOH- 


OH   OH    H 


CHOH- 


-C=C— OH 


OH   OH    H      OH 

Common  1-2  dienol  of  D-glucose,  D-mannose  and 
D-fructote. 


This  1,  2  dienol,  by  taking  up  one  molecule  of  water  at  the  double  bond 
and  rearrangement  goes  into  either  d-glucose,  d-mannose,  or  into  d-fructose, 
according  as  to  whether  the  OH  of  the  HOH  attaches  to  carbon  atom  1  or  2. 
In  this  manner  the  interconversions  of  the  hexoses  above  referred  to  are 
explained,  the  same  principles  holding  for  the  other  sugars  as  well  as  the 
d-glucose. 

In  a  similar  way  there  are  formed : 


OH       OH 


H 


CHaOH- 


H2OH, 


CHaOH- 


OH       OH 

2.3  Dienol. 


=  C  — C  — CHaOH 


OH    OH  OH  OH 

3.4  Dienol. 


These  dienols  are  also  capable  of  rearrangement  to  form  other  mono- 
saccharides  and  polysaccharides,  as  just  indicated.  However,  the  dienols 
are  capable  of  another  and  more  drastic  change.  As  is  almost  general  in 
carbon  compounds,  double  carbon  bonds  break  easily  and  are  highly  reactive. 
The  enols  break  apart  spontaneously  at  the  double  bonds ;  this  results  in  the 
formation  of  substances  of  very  great  reactivity,  capable  of  intramolecular 
rearrangement,  action  upon  each  other,  and  (if  there  is  oxygen  present),  the 
formation  of  the  large  variety  of  acids  briefly  described  above.  Thus,  e.  g.f 


INTRODUCTORY  DISCUSSION.  19 

the  breaking  of  the  1,  2  hexose  dienol  and  subsequent  immediate  oxidation 
would  result  in  the  formation  of  formic  and  d-arabonic  acids.  All  these 
actions  proceed  simultaneously,  the  final  products  depending  upon  the 
conditions  favoring  the  one  or  the  other.  By  vigorous  oxidation,  as  for 
instance  the  use  of  copper  hydroxide  or  silver  oxide,  the  products  of  the  more 
complete  dissociation  are  oxidized  to  form  the  corresponding  acids,  while 
with  air  or  hydrogen  peroxide  the  final  products  formed  are  from  the 
immediate  oxidation  of  the  dissociated  sugar  molecule. 

On  account  of  their  great  reactivity  it  has  not  been  possible  as  yet  to 
determine  definitely  the  composition  of  all  the  fragments  resulting  from 
the  dissociation  of  the  sugars.  The  principle,  however,  of  salt  formation 
and  subsequent  dissociation  seems  well  established.  Of  greatest  importance 
to  the  problem  of  metabolism  is  the  fact  that  precursory  to  the  oxidation  of 
sugars  is  the  dissociation  or  rearrangement  of  the  molecule  into  a  large 
number  of  pieces  of  high  reactivity  and  that  these  products  of  dissociation 
are  capable  of  the  most  multifarious  reactions  depending  upon  various  con- 
ditions, such  as  concentration,  temperature,  and  the  presence  of  oxygen. 

Prom  a  survey  of  the  phenomena  of  oxidation  over  the  very  extensive 
range  of  chemical  substances,  it  becomes  evident  that  a  primary  dissocation 
is  conditio  sine  qua  non  before  oxidation  can  take  place.  This  conception  in 
one  form  or  another  is  of  greatest  importance  to  the  physiologist  in  the 
interpretation  of  phenomena  of  catabolism,  but  has  been  considered  very 
little  by  plant  physiologists.  That  pure  aqueous  solutions  of  sugars  are 
relatively  stable  has  led  to  many  misconceptions  and  the  formulation  of 
elaborate  hypotheses  as  to  the  process  and  agents  inducing  glycolysis.  It 
need  hardly  be  mentioned  that  conditions  in  the  cell  are  very  different,  that 
the  reactions  do  not  take  place  in  pure  water  solution,  but  largely  in  a 
colloidal  mixture  in  the  presence  of  a  large  variety  of  inorganic  salts,  and 
that  such  conditions  lead  to  a  dissociation  of  the  sugar  molecule.  The  role 
and  mode  of  function  of  enzymes  is  in  all  probability  a  special  form  of  this 
group  of  reactions.  In  fact,  it  seems  doubtful  that  for  these  reactions,  at 
least,  it  is  necessary  to  assume  that  the  sugars  are  first  organized  into  a  large 
"  biogen  "  molecule  in  the  cell  prior  to  the  release  of  energy  or  oxidation. 
Such  an  assumption  lies  quite  outside  of  the  range  of  scientific  investigation. 

By  means  of  the  treatment  with  calcium  salts  of  organic  acids,  the 
products  of  dissociation  of  the  dextrose  molecule  are  capable  not  only  of 
rearrangement  and  oxidation,  but  also  of  union  to  form  polysaccharides  and 
more  complex  substances.1 

The  relatively  simple  dissociation  of  sugars  by  means  of  alkali  must 
yield  considerable  energy,  as  Nef  observed  a  decided  rise  in  temperature 
on  mixing  the  solutions."  It  would  be  exceedingly  interesting  to  have 
accurate  experimental  data  on  this  point. 

The  questions  relative  to  the  mechanism  of  the  utilization  of  energy  in 
the  vegetable  organism  have  as  yet  not  been  stated  by  plant  physiologists 

*  NEF,  J.  U.    Liebig's  Annalen,  403,  226-234,  373,  1913.        » Ibid.,  376,  12,  1910. 


20  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

in  such  terms  as  to  permit  of  any  well-defined  experimental  investigations. 
The  form  in  which  energy  is  utilized  in  the  plant  is  still  a  matter  of  much 
conjecture.  This  is,  of  course,  aside  from  the  utilization  of  solar  energy  in 
photosynthesis. 

Many  animal  physiologists  have  assumed  for  a  long  time  that  dextrose 
is  not  burned  as  such  in  the  body,  but  that  by  the  action  of  protoplasm  it  is 
first  dissociated  (in  a  very  similar  manner  as  has  been  described  through  the 
action  of  alkali)  to  form  various  dissociation  products,  which  in  turn  are 
either  oxidized  or  are  reduced  or  condensed  to  form  other  metabolic 
products.1  These  opinions  were  greatly  influenced  by  the  work  of  G.  von 
Liebig,  who  showed  that  the  legs  of  frogs  continued  to  react  and  produce 
CO2  in  an  oxygen-free  atmosphere,  and  by  that  of  Pettenkofer  and  Voit, 
who  demonstrated  that  the  total  protein  dissociation  is  independent  of 
oxygen.  Yoit  showed  that  for  proteins  the  metabolism  was  not  proportional 
to  the  oxygen  supply,  that  metabolism  was  a  process  of  cleavage  of  sugar, 
fat,  or  protein  molecules  into  simpler  products  which  then  united  with 
oxygen ;  in  brief,  that  absorption  of  oxygen  is  not  the  cause  of  metabolism 
but  rather  that  metabolism,  the  process  of  dissociation,  determines  the 
amount  of  oxygen  which  is  used.  This  whole  conception  has  been  tersely 
stated  by  Lusk :  "  Metabolism  vivifies  the  energy  potential  in  chemical 
compounds." ' 

That  the  production  of  heat  is  roughly  proportional  to  the  oxygen-supply 
has  been  a  familiar  fact  since  the  well-known  researches  of  Garreau  and  of 
Erikksson.  That  many  plants  live  and  grow  to  some  extent  for  a  limited  time 
in  an  atmosphere  freed  of  oxygen  is  also  a  matter  of  common  knowledge. 
Many  animals  exhibit  the  same  phenomenon,  although  to  a  more  limited 
extent  Under  these  conditions  the  organism  obtains  its  energy  through 
so-called  intra-molecular  respiration.  In  the  higher  plants  and  animals 
anoxybiosis  usually  produces  substances  which  are  toxic  to  the  organism, 
and  in  most  cases  suffocation  is  the  result  of  the  accumulation  of  these 
substances  to  an  extent  causing  death.  The  fact  that  in  general  plants  are 
more  resistant  to  these  effects  can  probably  be  explained  by  their  high 
synthetic  power.  Thus,  for  example,  alcohol  and  many  hydroxyl  acids  are 
converted  into  substances  which  can  be  drawn  into  the  stream  of  anabolism, 
especially  in  the  light. 

From  observations  on  the  temperature  rise  in  the  fermentation  of  sugars 
by  yeasts,  Fitz  comes  to  the  conclusion  that  each  gram  molecule  of  dextrose 
fermented  yields  57,500  calories.  The  heat  of  combustion  of  dextrose  is 
684,000  calories.*  These  figures  give  some  indication  of  the  cause  of  the 
differences  in  the  thermal  relations  of  the  two  processes  of  oxybiosis  and 
anoxybiosis.  However,  it  is  an  open  question  whether  the  thermal  prop- 
erties alone  express  the  true  energy  relations  in  so  complex  a  process  as 

*  Von,  C.  VON.    Handbuch  der  Physiologie  des  gesammtstoffwechsels  under  der  Fort- 

pflanzung,  6,  279, 1881.     Leipzig. 

*  LTTSK,  GRAHAM.     The  elements  of  the  science  of  nutrition,  page  33,  1917. 

H.    Grundlagen  und  Ergebnisse  der  Pflanzenchemie,  II,  page  161,  1909. 
Braunschweig. 


INTRODUCTORY  DISCUSSION.  21 

respiration.  Undoubtedly  the  economic  coefficient  varies  greatly  in  dif- 
ferent plants,  and  in  all  probability  heat  is  to  a  large  measure  only  the 
unavoidable  accompaniment  of  catabolism,  and  the  plant  must  use  other 
forms  of  energy  for  the  synthesis  of  formative  material.  The  results  of  the 
very  extensive  and  thorough  work  which  has  been  done  in  the  field  of 
mammalian  physiology  on  this  subject  as  yet  find  very  little  application  to 
the  problems  in  the  plant.  This,  of  course,  is  not  surprising  in  view  of  the 
fundamental  differences  in  the  normal  mode  of  life  and  functions  between 
plants  and  animals. 

There  is  as  yet  no  definite  way  of  determining  the  form  in  which  energy 
is  released  and  utilized  in  the  processes  in  the  living  plant  Practically  the 
only  factors  which  so  far  have  been  within  the  range  of  experimental  investi- 
gation have  been  the  study  of  the  end-results  and  outer  manifestations  of 
the  energy  transformations.  For  the  majority  of  plants  the  liberation  of 
heat  is  probably  the  most  general.  The  very  interesting  experiments  of 
Bonnier1  indicate  that  this  varies  greatly  under  various  conditions  and 
stages  of  development  By  comparing  the  heat  liberated  with  the  theoretical 
amount  possible  by  simple  combustion  calculated  from  the  absorption  of 
oxygen  and  liberation  of  carbon  dioxid,  Bonnier  found  that  the  former 
often  greatly  exceeds  the  latter,  while  in  other  cases,  as  in  mature  plants, 
the  heat  liberated  was  decidedly  lower  than  the  amount  calculated  from 
their  respiration.  While  these  results  but  indicate  the  transformations  of 
the  obscure  chemical  energy,  it  would  be  highly  desirable  to  elaborate 
investigations  of  this  nature,  and  to  do  so  in  collaboration  with  a  study  of 
the  energy  relations  of  the  intricate  chemical  dissociations  discussed  above. 
Investigations  thus  far  undertaken  have  not  contributed  greatly  to  a  fuller 
understanding  of  the  subject,  as  in  the  measurement  of  heat  liberated 
little  attention  has  been  paid  to  the  chemical  changes  and  the  nature  of 
exothermic  reactions  involved. 

Of  interest  in  this  connection  are  the  old  observations  of  Radzisezewski  * 
who  studied  the  phosphorescence  which  is  produced  in  plants  and  animals 
and  which  also  accompanies  the  autoxidation  of  a  very  large  number  of 
organic  compounds.  He  interpreted  his  findings  on  the  basis  of  the  old 
Schonbein  theory  of  oxidation,  and  showed  that  all  substances  which  by 
oxidation  Schonbein  had  noticed  formed  hydrogen  peroxide  and  active 
oxygen  also  produced  light,  and  generally  at  ordinary  temperatures. 
Eadzisezewski  was  the  first  to  observe  that  sodium  hydroxide  induces  this 
phosphorescence  in  many  organic  compounds ;  e.  g,,  all  aldehydes  and  their 
ammonia  derivatives,  the  higher  alcohols  (it  has  since  also  been  observed 
with  sugars),  fats,  the  higher  fatty  acids,  the  soaps,  and  a  large  variety  of 
others.  Knowledge  of  the  liberation  of  other  forms  of  energy  besides  heat 
in  the  dissociation  and  oxidative  reactions  induced  by  catalysts  may  be  of 

*BONNIEB,  G.    Recherches  sur  la  chaleur  v6g6tale,  Ann.  Science  Nat.  Serie,  VII, 

18,  1,  1893. 
'RADZISEZEWSKI,  M.     Ueber  der  Phosphoriscenz  der  organischen  und  der  organi- 

sierten  Koerper,  Lieblg's  Ann.  der  Chem.,  203,  305-336,  1881. 


22  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

the  utmost  importance  in  the  study  of  the  problem  of  metabolism  in  the 
plant. 

The  special  attention  which  has  been  given  here  to  results  so  far  obtained 
from  the  investigations  of  the  reactions  of  sugars  in  combination  with  salts 
and  alkalies  seem  warranted  in  that  they  represent  a  system  in  dynamic 
equilibrium;  the  breaking  down  is  always  accompanied  by  the  synthesis 
of  more  complex  substances,  and  while  such  a  complex  system  is  still  beyond 
the  experimental  control  of  physical  chemical  methods,  it  simulates  the 
dynamic  nature  of  living  things,  it  emphasizes  process  rather  than  single 
substances,  and  brings  these  processes  within  the  range  of  experimental 
science  by  centering  energy  changes  on  the  fundamental  properties  of  the 
carbon  atom.  Furthermore,  these  reactions  give  a  clear  indication  of  the 
nature  of  one  form,  at  least,  of  enzyme  action  in  a  homogeneous  system  and 
argue  strongly  for  the  theory  of  the  formation  of  intermediate  compounds. 

But  before  such  a  chemical  system  can  be  rationally  applied  to  the  f unc- 
tionings  of  a  living  plant,  some  further  knowledge  is  essential  of  the  nature 
of  the  carbohydrates  and  the  transformations  which  these  undergo  in  the 
plant  under  various  conditions.  Here  again  the  aspect  of  a  dynamic 
equilibrium  is  evident.  It  is  a  matter  of  common  knowledge  that  most 
plants  contain  monosaccharides,  disaccharides,  and  the  condensation  prod- 
ucts of  these  (the  polysaccharides)  in  various  proportions.  Each  of 
these  types  of  carbohydrates  is  what  might  be  termed  a  "  physiological 
group,"  and  the  proportion  in  which  they  are  present  in  a  plant  leaf, 
for  instance,  is  the  result  of  certain  factors,  and  in  turn  controls  to  a  large 
measure  the  physiological  activity  of  the  organism.  An  effort  has  been 
made  to  determine  some  of  the  conditions  affecting  this  equilibrium  of  mono- 
saccharide^l^disaccharide^Z^polysaccharide  and  to  study  the  effect  of  this 
on  metabolism. 

In  general,  chemical  inversion,  or  the  transformation  of  the  condensed 
to  the  simpler  molecules  capable  of  oxidation,  translocation,  and  the  forma- 
tion of  other  substances,  takes  place  under  conditions  of  ample  water-supply. 
However,  these  reversible  enzymatic  reactions  never  run  entirely  in  one 
direction ;  only  differences  between  the  two  are  observable.  We  are  dealing 
with  delicate  compound  dynamic  equilibria  involving  probably  dozens  of 
steps  and  many  more  substances.  The  enormous  importance  of  water  in 
various  functions  of  the  organism,  such  as  growth,  has  long  been  recognized. 
When  it  is  realized  that  it  is  to  a  great  extent  only  under  conditions  of 
sufficient  water  that  the  simpler  and  more  reactive  carbohydrates  are 
present  in  sufficient  amounts  in  the  cell,  this  fact  becomes  understandable. 
It  has  been  found  that  variations  in  the  water-content  of  the  cactus  joints 
greatly  influence  the  proportion  of  the  various  groups  of  sugars.  As  a 
consequence,  the  course  of  metabolism  is  also  decidedly  affected.  This 
undoubtedly  also  is  true  for  other  plants.  With  high  water-content  the 
polysaccharides  are  converted  into  monosaccharides,  and  from  all  evidence 
available  under  normal  conditions,  the  latter  are  the  carbohydrates  most 


INTRODUCTORY  DISCUSSION.  23 

readily  dissociated  and  drawn  into  the  stream  of  catabolism.  It  is  only 
under  conditions  of  stress  that  the  plant  uses  the  disaccharides  or  poly- 
saccharides  with  the  result  of  the  formation  of  pentosans  and  pentoses. 
These  actions  will  be  discussed  under  the  section  on  pentose  sugars.  With 
ample  water-supply  and  oxygen  the  hexoses  are  burned  in  such  a  manner 
that  there  is  not  an  accumulation  of  end-products  which  are  deleterious  to 
the  plant.  Another  feature  of  such  complete  burning  is  the  influence  of  the 
products  on  the  water-absorbing  capacity  of  the  colloidal  substratum  of  the 
cell,  and  hence  on  the  growth  of  the  organism.  The  influence  of  the  various 
intermediate  products  of  metabolism  on  imbibition  and  the  manner  in  which 
this  is  correlated  with  growth  is  being  extensively  studied  at  the  Desert 
Laboratory. 

Early  in  the  course  of  the  investigations  on  the  metabolism  of  succulent 
plants  it  was  recognized  that  the  organic  acids  found  therein  are  the  accumu- 
lating intermediate  or  end-products  of  a  modified  form  of  catabolism  due  to 
morphological  peculiarities  of  the  plant  The  chemical  principle  which 
underlies  the  acidification  is  the  formation  of  acids  by  the  oxidation  of 
sugars ;  the  manner  in  which  this  occurs  and  the  products  formed  have 
already  been  indicated.  One  of  the  principal  agents  of  deacidification  in 
plants  is  sunlight ;  organic  acids,  such  as  oxalic,  malic,  tartaric,  etc.,  break 
down  very  readily  in  the  light  in  that  the  carboxyl  group,  COOH,  splits  off 
COj.1  It  is  a  noteworthy  fact,  discussed  by  Richards,  that  cacti  rich  in  acid 
exhale  carbon  dioxid  abundantly  in  the  sunlight,  even  at  moderately  low 
temperatures.  This  fact  also  finds  application  in  the  formation  of  pentose 
sugars,  as  will  be  discussed  later. 

The  total  carbohydrate  content  or  of  food-supply  in  general  is  of  little 
significance  or  value  in  studying  the  various  functions  of  a  plant  It  is 
rather  the  nature  of  the  sugars,  or  the  degree  of  general  chemical  inversion, 
that  determines  the  supply  of  material  available  for  respiration  or  growth. 
The  relative  amounts  and  proportion  of  the  carbohydrates  in  a  leaf,  for 
example,  are  dependent  upon  several  factors,  primarily  water-content  and 
temperature.  Also  it  is  essential  to  realize  that  the  transformation  of  the 
various  groups  of  carbohydrates  represents  thus  an  equilibrium  system 
subject  to  change  by  several  factors.  It  is  evident,  therefore,  that  only 
after  determining  the  influence  of  these  factors  and  taking  them  into  con- 
sideration can  any  safe  conclusion  be  drawn  as  to  the  significance  of  the 
presence  of  any  one  of  the  various  groups  of  sugars.  This  fact  finds 
immediate  application  in  the  problem  of  photosynthesis.  There  has  been  a 
great  deal  of  dispute  over  the  first  sugar  formed  in  the  process  of  photo- 
synthesis. Various  workers  have  arrived  at  conclusions  which  are  still  very 
contradictory.  Unfortunately,  in  most  of  the  work  the  factors  already 
referred  to  were  not  determined,  so  that  it  is  impossible  to  explain  the 
variable  results  on  this  basis. 

1  RICHARDS,  H.  M.    L.  c.,  83. 

SPOEHB,  H.  A.     Photochemische  Vorgaenge  bei  der  Diurnalen  entsaeurung  der 
Succulenten,  Biochem.  Zeitschr.,  57,  95-111,  1913. 


24  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

II.  HISTORICAL. 

Beyond  the  proximate  analyses  incident  to  investigations  of  the  food 
value  of  cacti  for  agricultural  purposes,  very  little  work  has  appeared  on 
the  carbohydrates  of  the  cacti.1  Nor  is  there  any  extensive  information  on 
the  subject  available  for  other  succulent  plants.  However,  on  account  of 
the  complex  character  of  its  respiratory  processes,  its  structural  peculiari- 
ties, and  the  rather  extreme  climatic  conditions  under  which  many  of  its 
members  live,  this  group  of  plants  has  been  subjected  to  extensive  physio- 
logical investigations.  Some  of  the  earliest  and  now  classical  researches  on 
respiration  were  carried  out  with  this  material. 

The  interest  has  centered  primarily  around  the  gas  interchange  and  the 
periodic  acidification  and  deacidification  of  the  plants.  De  Saussure '  was 
the  first  to  study  thoroughly  the  respiratory  relations  of  these  plants.  The 
question  of  acidity  in  succulents  has  been  subjected  to  extensive  investiga- 
tions ;  most  recent  are  the  very  thorough  researches  of  Richards  *  and  those 
of  Jenny  Hempel.4  In  the  publication  of  Richards  is  given  a  clear  discus- 
sion of  the  historical  development  of  the  subject,  and  the  conditions  induc- 
ing acidification  and  deacidification,  together  with  the  effect  on  the  respira- 
tory quotient,  are  there  elaborately  formulated,  based  upon  wide  observation 
and  comprehensive  experimentation. 

The  relation  of  the  plant  acids  to  the  carbohydrates  has  been  the  subject 
of  much  speculation  ever  since  the  appearance  of  Liebig's  theories  of  the 
origin  of  sugars  from  hydroxy  acids  in  ripening  fruits.  Although  the 
weight  of  evidence  seems  to  be  against  the  Liebig  theory,  and  it  is  con- 
sidered as  untenable  by  most  plant  physiologists,  the  principle  thereof  has 
been  frequently  revived  and  has  found  expression  in  modified  forms.*  The 
formation  of  organic  acids  as  catabolic  products  of  carbohydrate  metabo- 
lism has  received  very  little  attention  from  plant  physiologists.  From  the 
chemical  evidence  available,  many  of  the  intermediate  products  of  sugar 
degradation  are  acids  of  the  nature  of  those  found  in  the  plant,  as  has  been 
discussed  in  the  introduction.  However,  the  manner  in  which  the  carbo- 
hydrates are  first  affected,  the  actual  mode  of  acid  formation  in  the  plant, 
and  the  further  transformations  are  all  questions  toward  which  very  little 
physiological  inquiry  has  been  directed.  As  prerequisite  knowledge  for 
investigation  of  these  problems,  we  must  have  some  understanding  of  the 
interrelation  and  transformations  of  the  carbohydrates  from  the  simple  to 
the  complex,  and  vice  versa,  the  conditions  inducing  such  changes  and  also, 

1  GBOTITH,  D.,  and  R.  F.  HARE.    Prickly  pear  and  other  cacti  as  food  for  stock.    Bull. 
60,  New  Mexico  College  of  Agriculture  and  Mechanic  Arts,  1906.    Ibid.,  72. 
HABBINGTON,  H.  H.    Analysis  of  cactacus.    Tex.  Expt.  Station  Report  1,  28, 1888. 

*  DE  SAUSSUBE,  THEO.    Recherche  chemique  sur  la  v6g6tation.     1804. 
•RICHABDS,  H.  M.     Acidity  and  gas  interchange  in  cacti.     Carnegie  Inst  Wash. 

Pub.  No.  209,  1915. 

*  HEMPEL,  JENNY.    Buffer  processes  in  the  metabolism  of  succulent  plants.    Comptes- 

rendus  des  travaux  du  Laboratoire  de  Carlsberg,  13,  1917. 

*  BATO,  E.    Ueber  die  Genesis  der  Kohlenhydrate.    Naturwissenschaften  1,  474-477, 

1913.    Zeit.  physik  chem.,  63,  683-710. 


HISTORICAL.  25 

of  course,  the  chemical  nature  of  the  carbohydrates  in  the  organism  under 
investigation. 

For  the  succulent  plants  especially  very  little  data  of  this  nature  have 
been  gathered.  In  1902  Harlay1  published  some  investigations  on  the 
mucilage  of  Opuntia  vulgaris.  The  mucilaginous  substances  are  located  in 
special,  large  cells.  These  were  made  visible  in  material  which  was  con- 
served in  alcohol  and  examined  with  lead  subacetate.  By  allowing  the 
cactus  joints  to  remain  in  an  atmosphere  of  ether,  Harlay  found  that  the 
mucilages  exuded  as  a  clear,  slightly  colored,  thick  liquid.  He  obtained  the 
mucilage  by  removing  the  exterior  chlorophyllous  portion,  chopping,  grind- 
ing with  sharp  sand,  heating  with  water  at  110°,  filtering  through  cloth, 
and  precipitating  with  90  per  cent  alcohol ;  this  precipitate  was  filtered  off, 
washed  with  alcohol  and  dried.  Thus  he  obtained  a  gray  powder,  which 
when  dissolved  in  water  did  not  reduce  Fehling's  solution,  but  always  con- 
tained a  small  amount  of  inorganic  material.  The  aqueous  solution  could 
not  be  filtered.  His  analysis  indicated  the  presence  of  galactose  and 
arabinose. 

Eecently  Long*  made  some  analyses  of  the  carbohydrate-content  of 
Echinocadus  in  collaboration  with  the  studies  of  MacDougal  on  the  water 
balance  and  desiccation  of  this  plant. 

1  HABLAY,  V.    Sur  le  mucilage  du  cactus  a  raquettes,  Opuntia  vulgaris  Mil.    Jour,  de 

Pharm.  et  de  Chem.  (6),  16,  193-198,  1902. 
'MAcDouoAL,  D.  T.,  B.  R.  LONG,  and  J.  G.  BBOWN.    End  results  of  desiccation  and 

respiration  in  succulent  plants.    Physiol.  Researches  1,  289-325,  1915. 
LONG,  E.  R.    Further  results  in  desiccation  and  respiration  of  Echinocactus.    Bot. 

Gaz.,  65,  334-358,  1918. 


26  THE   CARBOHYDEATE  ECONOMY  OF   CACTI. 

III.  EXPERIMENTAL  METHODS. 
SELECTION  AND  PREPARATION  OF  MATERIAL. 

The  greater  part  of  the  work  here  recorded  was  done  with  Opuntia  phcea- 
cantha,  known  locally  as  Opuntia  blakeana.1  Other  species  were  also  used, 
but  the  results  do  not  differ  materially  from  those  obtained  with  this  plant. 
In  working  with  the  cacti  considerable  difficulty  is  encountered  on  account 
of  the  large  amount  of  mucilaginous  substances  therein.  However,  this  in 
itself  is  a  subject  of  great  interest,  the  nature,  source,  and  function  of  these 
slimes  being  still  unknown  and  to  a  greater  or  less  extent  forming  a  char- 
acteristic property  of  all  succulent  plants.  Furthermore,  the  joints  of 
Opuntia  phceacaniha  are  of  a  size  that  can  be  most  easily  handled  for  the 
various  experimental  procedures,  and  also  grow  in  abundance  on  the  labora- 
tory domain.  On  account  of  the  smaller  amount  of  mucilaginous  substances, 
Opuntia,  versicolor 2  was  used  where  it  was  necessary  to  press  out  the  juice. 
Such  a  procedure  is  quite  impossible  with  Opuntia  phceacaniha,  especially 
during  the  dry  seasons. 

Each  series  of  determinations  or  experiments  was  carried  out  with 
material  from  one  plant  Although  there  are  naturally  differences  among 
individual  plants,  the  changes  which  these  undergo  with  season  and  under 
experimental  conditions  are  entirely  parallel.  Large,  healthy  plants  have 
a  sufficient  number  of  joints  of  the  same  age,  so  that  extended  experiments 
can  be  conducted  with  material  from  the  same  plant  and  a  number  of  joints 
can  be  used  for  each  determination.  For  the  work  on  sugar  estimation  the 
material  was  collected  between  10  and  11  o'clock  in  the  morning.  The 
joints  were  removed  by  cutting  at  the  base  with  a  sharp  knife.  The  cut 
surfaces  were  2  to  3  square  centimeters,  and  no  loss  of  juice  or  other  injury 
was  ever  observed  therefrom.  The  joints  were  immediately  taken  to  the 
laboratory,  and  the  spines  were  removed  with  sharp  pruning  shears,  care 
being  taken  not  to  cause  any  injury. 

The  cacti  offer  splendid  material  for  investigations  of  this  nature.  All 
the  year  fresh  and  normal  material  can  be  obtained  in  abundance;  it  is 
notably  resistant  to  lack  of  water,  as  well  as  to  variations  in  temperature, 
and  can  be  kept  in  the  dark  for  months  without  fatal  results.  Nevertheless 
all  these  conditions  effect  decided  changes  in  the  plants.  Furthermore,  they 
can  be  easily  grown,  even  in  nutrient  solutions,  and  if  necessary  a  single 
joint  offers  ample  material  for  complete  analysis. 

PREPARATION  OF  MATERIAL  FOR  ESTIMATION  OF  SUGARS. 

All  material,  whether  it  had  been  subjected  to  experimental  conditions 
or  taken  immediately  from  out  of  doors,  was  prepared  alike  for  analysis. 
The  joints,  freed  from  the  spines,  were  cut  into  three  or  four  strips  and 

aBBiTTON,  N.  Li.,  and  J.  N.  ROSE.     The  Cactaceae:     Description  and  illustration  of 
plants  of  the  cactus  family.    Carnegie  Inst.  Wash.  Pub.  No.  248,  vol.  i,  p.  144. 
1  Ibid.,  p.  62. 


EXPERIMENTAL  METHODS.  27 

ground  in  a  meat-chopper.  By  using  a  fairly  coarse  cutter,  there  was  no 
loss  of  juice.  The  manipulation  was  carried  out  as  rapidly  as  possible. 
The  ground  mass  was  thoroughly  mixed  and  put  into  a  dish,  covered,  and 
placed  in  a  hot-air  oven  at  98°.  In  this  manner  the  plant  material  is  quickly 
killed  and  all  danger  from  enzyme  actions,  which  by  the  usual  means  of 
drying  are  first  considerably  accelerated,  is  thus  avoided.  On  account  of 
the  danger  of  subsequent  action  of  enzymes  on  the  carbohydrates,  expression 
of  the  juice  of  a  plant  is  also  impractical.  After  heating  thus  for  about 
30  minutes,  the  cover  was  removed  and  the  material  dried  for  24  hours  at 
98°.  It  was  then  ground  to  a  fine  homogeneous  powder  and  was  ready  for 
analysis. 

THE  DETERMINATION  OF  DRY  WEIGHT. 

As  has  been  indicated,  in  order  to  gain  a  clear  conception  of  the  carbo- 
hydrate transformations  in  a  vegetable  organism,  it  is  essential  to  know  the 
water  relations.  For  this  reason  each  sugar  analysis  was  supplemented 
with  a  determination  of  the  dry  weight.  A  small  amount  (about  10  grams) 
of  homogeneous  freshly  ground  material  was  placed  in  a  tared  wide-mouth 
weighing  bottle,  weighed,  first  heated  with  the  cover  on  as  described  above, 
and  then  dried  at  98°  and  cooled  in  a  desiccator  before  weighing.  This 
always  was  done  in  duplicate  or  triplicate  and  the  results  agreed  well  with 
each  other.  Further  heating  at  98°  or  keeping  in  a  desiccator  for  several 
days  caused  no  further  appreciable  loss  in  weight.  Separate  experiments 
showed  that  in  this  drying  process  the  disaccharides  were  not  appreciably 
inverted. 

PREPARATION  OF  MATERIAL  FOR  ANALYSIS. 

A  separation  of  the  simpler  sugars  from  the  condensed  polysaccharides 
was  attained  by  extracting  one  portion  of  the  dry  material  with  95  per  cent 
alcohol  and  by  hydrolyzing  another  portion  with  dilute  acid.  For  the 
alcoholic  extract  30  to  50  grams  of  the  dry  material  was  mixed  with  2  to  5 
grams  of  powdered  calcium  carbonate  in  order  to  neutralize  the  plant  acids 
present  and  thus  prevent  inversion  of  the  disaccharides.  Although  calcium 
carbonate  gives  a  very  slightly  alkaline  solution,  this  is  not  sufficient  to  cause 
enolization  and  consequent  rearrangement  of  hexoses  or  formation  of 
saccharinic  acids.  Stronger  alkalies  are  to  be  avoided,  as  they  cause  the 
reactions  mentioned.  This  is  a  very  serious  source  of  error  in  much  of  the 
work  in  the  literature  on  the  nature  of  hexoses  in  leaves,  as  apparently  the 
ease  of  mutual  rearrangement  of,  e.  g.,  d-glucose,  d-mannose,  and  d-levulose 
was  entirely  disregarded.  These  transformations  have  already  been  dis- 
cussed in  the  introductory  discussion. 

For  the  present  purpose  no  attempt  was  made  to  distinguish  these  sugars 
in  the  analyses,  but  they  are  regarded  rather  as  belonging  to  one  physio- 
logical group.  The  calcium  salts  of  most  of  the  plant  acids  are  quite 
insoluble  in  alcohol.  The  mixture  of  the  dry  plant  material  and  calcium 
carbonate  was  extracted  twice,  with  10  times  its  weight  of  95  per  cent 


28  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

alcohol,  each  time  heating  to  boiling  for  3  hours,  filtered,  and  the  residue 
washed  with  hot  alcohol.  This  procedure  removed  all  alcohol-soluble 
sugars.  The  alcohol  was  then  distilled  off  at  reduced  pressure,  a  greenish- 
brown  gum  remaining.  To  this  was  added  hot  water  to  dissolve  the  sugars ; 
a  considerable  amount  of  oil  (largely  chlorophyll)  was  insoluble.  A  small 
quantity,  0.2  gram,  of  blood  charcoal  greatly  facilitated  the  filtration.  With- 
out the  addition  of  the  blood  charcoal  it  was  almost  impossible  to  get  a  clear 
solution  on  account  of  the  clogging  of  the  filter-paper  by  the  fine  oil  particles. 
In  order  to  make  negligible  any  loss  of  sugar  by  adsorption  by  the  charcoal, 
the  same  lot  of  Merck's  blood  charcoal  was  used  throughout.1 

As  the  amount  of  protein  and  tannin  in  the  cactus  is  very  small,  it  was 
found  inadvisable  to  clarify  the  sugar  solutions  with  lead  acetate.  The 
water  from  the  clear  aqueous  solution  was  distilled  off  at  reduced  pressure 
(75°  and  35  mm.)  in  a  tared  flask,  finally  with  the  water-bath  boiling. 
The  residual  light-brown  gum  was  weighed,  dissolved  in  water,  and  the 
solution  made  up  to  a  definite  volume.  The  sugars  were  then  determined 
as  described  below.  The  reducing  sugars  present  were  monosaccharide 
hexoses  and  pentoses.  The  mixture  was  then  hydrolyzed  with  hydrochloric 
acid,  using  5  c.  c.  normal  acid  solution  for  every  100  c.  c.  of  the  sugar  solu- 
tion and  heating  for  3  hours  at  80°.  Repeated  trials  with  longer  heating 
showed  that  the  inversion  was  complete.  By  the  use  of  invertine,  the  same 
values  of  cupric  reduction  were  obtained  as  with  hydrochloric  acid,  indi- 
cating that  there  was  no  maltose  present 

The  mixture  was  neutralized  carefully  with  sodium  bicarbonate,  made  up 
to  a  definite  volume,  and  the  sugars  determined.  This  determination  repre- 
sents monosaccharide  hexoses  and  pentoses  and  invert  sugar.  The  sugar 
solution  was  then  fermented  in  order  to  remove  all  hexose  sugars.  For  this 
a  fresh  preparation  of  carefully  washed  bakers'  yeast  was  used.  To  the 
sterilized  sugar  solution  was  added  the  yeast,  mixed  with  distilled  water  to 
a  thin  paste,  0.25  gram  pressed  yeast  per  gram  of  original  gum.  The  vessel 
was  stoppered  with  cotton  and  left  at  30°  to  35°  for  30  to  40  hours.  Sepa- 
rate experiments  with  various  mixtures  of  invert  sugar  and  1-arabinose  and 
1-xylose  showed  that  by  this  procedure  all  hexose  sugars  present  in  the  plant 
were  removed,  while  the  pentoses  remained  unchanged.  The  mixture  was 
then  filtered  and  distilled  at  reduced  pressure  in  order  to  remove  the  prod- 
ucts of  fermentation.  The  residual  gum  was  dissolved  in  a  definite  volume 
of  water  and  the  sugars  determined  with  the  alkaline  copper  solution. 

For  the  estimation  of  the  polysaccharides,  5  to  15  grams  of  the  dry 
material  was  hydrolyzed  with  200  to  600  c.  c.  of  1  per  cent  hydrochloric 
acid  in  a  flask,  with  reflux  condenser,  in  the  boiling-water  bath  for  3  hours. 
The  mixture  was  filtered,  washed  thoroughly  with  hot  water,  the  filtrate 
neutralized  carefully  with  sodium  bicarbonate,  and  made  up  to  a  definite 

1  WOODYATT,  R.  T.,  and  H.  F.  HELMHOLTZ.    The  use  of  blood  charcoal  as  a  clearing 
agent  for  urine  containing  glucose.    Arch.  Intern.  Med.,  7,  598-601,  1911. 


EXPERIMENTAL  METHODS.  29 

volume.  The  solution  was  then  ready  for  analysis.  These  solutions  were  a 
light-brown  color;  they  were  not  treated  either  with  lead  acetate  or  blood 
charcoal,  as  the  amount  of  error  produced  by  such  treatment  is  equal  to  or 
even  greater  than  that  produced  by  the  very  small  amount  of  protein  or 
tannin  substances  in  the  original  material.  The  amount  of  sugar  here 
found  was  equal  to  the  hydrolyzed  alcoholic  extract  plus  the  amount  of 
sugar  in  the  residue  from  the  alcoholic  extraction  when  the  former  was 
hydrolyzed  and  treated  in  a  similar  manner.  The  residue  from  the  alcoholic 
extraction  contained  a  slight  excess  of  calcium  carbonate.  It  also  had  to 
be  entirely  freed  from  alcohol  before  it  could  be  used  for  the  estimation  of 
polysaccharides.  For  these  and  several  other  minor  reasons,  it  was  found 
much  more  rapid  to  make  these  determinations  with  a  fresh  sample  of  the 
dry  material  than  with  the  residue  from  the  alcoholic  extraction,  and  it  was 
found  that  the  polysaccharides  could  be  determined  accurately  from  the 
total  sugars  by  calculation. 

One  per  cent  hydrochloric  acid  was  decided  upon  for  the  hydrolysis  after 
considerable  experimentation.  It  was  desired  to  hydrolyze  to  monosac- 
charides  all  those  disaccharides  and  polysaccharides  including  starch,  dex- 
trine and  the  mucilaginous  substances,  which  may  be  important  to  the  plant 
as  reserve  food  material,  but  without  affecting  the  cellulose  which  goes  to 
make  up  the  walls  and  vessels  of  the  plant.  It  was  necessary,  therefore,  to 
utilize  the  lowest  concentration  of  acid  which  still  hydrolyzed  completely 
the  normal  reserve  materials.  Hydrochloric  acid  affects  cellulose  less  than 
do  other  mineral  acids  of  the  same  ionic  concentration.  It  was  found  that 
when  the  acid  mixture  was  heated  for  3  hours  on  the  boiling-water  bath  a 
rather  definite  end-point  had  been  reached ;  further  prolonged  heating  and 
using  fresh  acid  caused  exceedingly  slight  increase  in  the  reducing  power  of 
the  hydrolyzed  mixture.  Higher  concentrations  of  acid  already  seemed 
to  affect  the  cellulose  so  that  no  such  definite  end-point  was  obtained ;  while 
still  lower  concentrations  required  considerably  longer  heating  in  order  to 
completely  hydrolyze  the  starch  and  other  polysaccharides.  There  was 
always  an  odor  of  furfural  in  the  hydrolyzed  mixture,  but  this  was  very 
slight,  so  that  the  pentose  values  were  probably  affected  but  very  little. 
With  higher  concentrations  of  hydrochloric  acid,  the  furfural  was  much 
more  noticeable. 

Judging  from  the  microchemical  test  with  iodine,  the  platyopuntias  con- 
tain an  abundance  of  small  starch-grains.  When  the  dried  material  is 
mixed  with  water  it  takes  on  the  same  slimy  consistence  of  the  freshly- 
ground  cactus.  It  was,  therefore,  impossible  to  determine  the  starch 
chemically  by  the  use  of  taka-diastase  on  account  of  the  mucilaginous  sub- 
stances. These  can  not  be  filtered  through  filter-paper  even  after  the  addi- 
tion of  much  water,  and  boiling  the  solution  is  without  effect  on  them,  nor 
can  they  be  precipitated  except  by  the  use  of  large  quantities  of  alcohol 
The  clear  mucilage  which  can  be  obtained  by  the  use  of  a  filter-press  always 
contains  considerable  starch. 


30  THE  CABBOHYDBATE  ECONOMY  OF   CACTI. 

A  quantity  of  the  dried  cactus  material  was  mixed  with  water  and  boiled 
for  2  hours,  cooled,  a  solution  of  taka-diastase  added,  and  kept  at  37°  for 
22  hours.  This  procedure  was  repeated  for  8  days,  each  time  adding  fresh 
taka-diastase,  and  at  the  end  of  this  time  the  small  pieces  of  cactus,  after 
treatment  with  alcohol  and  iodine  in  the  usual  manner,  still  showed  dis- 
tinctly the  presence  of  starch.  The  slime  of  the  cactus  was  quite  unaffected 
by  this  treatment.  A  quantity  of  corn  starch  treated  in  a  similar  manner 
was  completely  hydrolyzed  after  the  first  day.  It  appears,  therefore,  that 
the  slime  in  some  manner  protects  the  starch  against  the  action  of  the  taka- 
diastase.  It  is  evident  that  plant  material  containing  such  mucilaginous 
substances  offers  some  unusual  difficulties  and  is  not  amenable  to  the  ordi- 
nary methods  of  analysis.  These  slimes  are  very  readily  hydrolyzed  by 
dilute  acids ;  by  such  treatment  they  lose  entirely  their  mucilaginous  char- 
acter and  the  solutions  can  be  easily  filtered. 

Cellulose  was  determined  by  taking  the  residue  from  the  acid  hydrolysis, 
digesting  it  with  1.25  per  cent  KOH,  washing  thoroughly,  and  filtering 
through  a  tared  filter-paper  and  drying.  The  dried  mass  was  then  incin- 
erated in  a  crucible  and  the  ash  subtracted  from  the  total  weight  of  the 
crude  fiber.  The  first  determinations  were  made  by  use  of  the  chlorination 
method,1  but  as  the  differences  between  this  and  the  much  simpler  method 
just  described  were  found  to  be  very  slight  and  within  the  experimental 
error  of  either  method,  the  first  procedure  was  followed,  which  yielded 
results  that  had  at  least  a  comparative  value. 

CALCULATION  OF  ANALYTICAL  DATA. 

The  analyses  were  calculated  in  terms  of  cubic  centimeters  of  standard- 
ized Fehlingfs  solution  reduced,  per  gram  of  dry  sample.  The  following 
deductions  represent  the  various  sugars.  In  the  solution  of  the  acid 
hydrolysis  the  copper  values  are  distributed  as  follows: 

A.  Total  sugars  of  the  hydrolyzed  material,  i.  e.,  the  monosaccharides  as  well  as 

the  hydrolyzed  polysaccharides. 

B.  Fermented  residue  of  A,  or  total  pentose  sugars,  including  monosaccharide 

pentoses  and  pentosans. 

In  the  alcoholic  extraction : 

C.  Monosaccharide  hexose  and  pentose  sugars. 

D.  Hydrolyzed  product  of  C  or  inverted  disaccharides,  original  hexose,  and 

pentose.    There  are  no  disaccharide  pentoses  present. 

E.  The  fermented  residue  of  D  or  monosaccharide  pentose. 

Then  the  copper  value  of  the  various  sugars  are : 

A.  Total  sugars.  C.  Monosaccharides. 

A-D.  Total  polysaccharides.  D-C.  Disaccharides. 

D-E.  Disaccharides  plus  hexoses.  C-E.  Hexoses. 

A-B.  Total  hexose  sugars.  B.  Total  pentoses. 

(A-B)-(D-E).  Hexose  polysaccharides.  E.  Monosaccharide  pentoses. 

B-E.  Pentosan. 

1  CROSS,  C.  F.,  and  E.  S.  BEVAN.    Cellulose.    Page  94,  1895.    London. 


EXPERIMENTAL  METHODS.  31 

The  reducing  power  of  these  sugars  is  not  equal,  but  the  reduced  copper 
was  apportioned  as  follows :  100  c.  c.  standarized  Fehling's  solution  =  1.00 
gram  d-glucose,  0.95  gram  invert  sugar,  0.90  gram  1-xylose,  0.90  gram 
hexose  polysaccharides,  and  0.85  gram  pentosan.  With  these  figures  the 
amount  of  the  various  sugars  per  gram  of  dry  material  and  in  some  cases 
of  original  fresh  material  was  calculated. 

THE  ANALYSIS  OF  THE  SUGAR  SOLUTIONS. 

On  account  of  the  large  number  of  estimations  of  sugar  which  had  to  be 
made,  it  was  necessary  to  employ  a  method  of  sugar  determination  which 
was  rapid  as  well  as  accurate.  The  application  of  the  reduction  of  alkaline 
copper  solutions  by  sugars — that  is,  some  modification  of  the  Fehling 
method — is  best  suited  to  this  purpose.  The  examination  of  the  various 
current  processes  showed  that  these  are  open  to  serious  error. 

In  the  following  method  an  attempt  has  been  made  to  modify  the  usual 
procedures  of  sugar  determinations  by  means  of  copper  in  such  a  manner 
as  to  avoid  most  of  the  sources  of  error  inherent  to  these,  especially  as 
applied  to  the  sugar  solutions  under  consideration.  It  will  not  be  necessary 
to  enter  into  a  detailed  discussion  of  the  theory  and  practice  of  sugar 
determinations  by  means  of  alkaline  copper  solutions.  Such  critical  and 
experimental  studies  have  been  carried  out  by  several  workers,  especially 
by  Pflueger1  and  by  Peters/  which  have  yielded  valuable  results,  but 
unfortunately  have  not  been  as  universally  applied  as  they  deserve. 

The  results  of  these  studies  indicate  clearly  that  where  rapidity  is  of 
importance  only  volumetric  methods  should  be  considered.  Any  doubt 
regarding  the  accuracy  of  the  volumetric  procedure  can  pertain  only  to  the 
measurement  of  the  amount  of  copper  reduced  by  the  sugar ;  the  conditions 
under  which  the  reduction  is  carried  out  must  in  any  case  be  carefully 
standardized.  The  majority  of  the  methods  require  a  filtration  of  the 
cuprous  oxide  which  is  either  weighed  as  such  or  dissolved,  and  then  deter- 
mined by  some  suitable  method. 

It  will  be  necessary  to  consider  but  a  few  of  the  most  important  factors 
affecting  the  accuracy  of  the  procedure.  The  filtration  of  the  cuprous  oxide 
formed  by  the  reduction  of  the  alkaline  copper  solution,  through  an  asbestos 
filter  or  some  similar  device,  is  open  to  several  sources  of  error.  Thus,  it 
has  been  found  by  several  investigators  that  the  weight  of  the  asbestos  filters 
is  not  constant  because  of  solution  of  the  asbestos  and  the  self -reduction  of 
the  reagents,  and  requires  a  correction  which  can  not  always  be  applied 
safely.  In  determining  the  sugars  in  solutions  obtained  from  plant  material 
such  as  the  cacti,  the  precipitated  cuprous  oxide  contains  contaminating 
substances  which  are  undeterminable  and  liable  to  affect  the  subsequent 

1  PFLUEGER,  E.    Untersuchungen  ueber  die  quantitative  Analyse  des  Traubenzuckers. 

Arch.  Ges.  Physiol.,  69,  399-471,  1898. 
*  PETEES,  A.  W.    A  critical  study  of  sugar  analysis  by  copper  reduction  methods. 

Jour.  Amer.  Chem.  Soc.,  34,  928-954,  1912. 


32 


THE   CARBOHYDRATE  ECONOMY   OF  CACTI. 


steps  if  the  cuprous  oxide  is  dissolved.  Furthermore,  the  filtration  must 
proceed  as  rapidly  as  possible  and  not  infrequently  do  these  contaminating 
substances  greatly  delay  this  process.  Thus  there  is  always  danger  of 
resolution  of  the  cuprous  oxide  in  the  alkaline  liquid,  especially  in  the 
presence  of  dissolved  atmospheric  oxygen.  It  was  therefore  desirable  to 
employ  a  method  which  did  not  depend  upon  the  determination  of  the 
cuprous  oxide  formed  in  the  reduction  and  which  also  obviated  the  necessity 
of  filtering  the  solution.  The  only  alternative  was  to  employ  the  residual 
method,  which  depends  upon  the  estimation  of  the  unreduced  copper.  The 
accuracy  of  such  a  procedure  naturally  depends  upon  a  good  method  of 
determining  copper.  This  has  been  worked  out  by  Peters  in  the  iodide 
method  and  yields  most  satisfactory  results. 

In  the  method  used  in  this  investigation,  the  reduction  is 
carried  out  in  specially  designed  tubes  (fig.  1)  of  a  capacity  of 
10  to  50  c.  c.,  depending  upon  the  amount  and  concentration  of 
the  sugar  solution  to  be  determined.  A  very  liberal  excess  of 
copper  over  that  required  to  oxidize  the  sugar  in  solution  is 
always  provided.  After  heating  a  definite  length  of  time,  the 
tubes  are  rapidly  cooled,  made  up  to  volume  with  distilled  water, 
shaken  in  order  to  mix  thoroughly,  and  then  centrif  uged  until  all 
the  cuprous  oxide  (and  other  solid  material)  is  in  a  compact  mass 
at  the  bottom  of  the  tube.  The  residual  copper  in  the  clear 
supernatant  solution  is  then  drawn  off  with  a  pipette  and  several 
determinations  are  made  by  the  iodide-thiosulphate  method.  The 
difference  between  this  result  and  the  original  strength  of  the 
copper  solution  represents  the  amount  of  copper  reduced  and  is 
a  quantitative  index  of  the  amount  of  sugar  originally  present. 
Experience  with  this  and  various  other  methods  of  sugar  deter- 
mination by  means  of  alkaline  copper  solutions  has  taught  that 
reliable  and  quantitatively  comparable  results  can  be  obtained 
only  by  means  of  a  most  careful  quantitative  standardization  of 
each  step  in  the  manipulation.  Hence  it  is  also  necessary  to  adhere  to  the 
same  procedure  throughout  an  investigation.  The  sugar  determinations 
were  made  by  use  of  Fehling's  solution  (Soxhlet's  modification:  69.28 
CuSO*  •  5H2O  per  liter),  although  it  was  later  found  that  a  solution  con- 
taining sodium  carbonate  instead  of  caustic  alkali  possessed  certain  advan- 
tages, because  the  weaker  alkali  affects  the  sugar  less  profoundly.  The 
Fehling's  solution  was  used  throughout  in  order  to  maintain  the  uniformity 
of  procedure.  Thus  solution  A  was  made  up  of  69.28  grams  of  pure 
CuSO4  •  5H2O  in  1,000  c,  c.  water,  while  solution  B  contained  346  grams 
of  sodium  potassium  tartrate  and  100  grams  of  sodium  hydroxide  in  1,000 
c.  c.  of  water.  As  will  be  seen  later,  a  correction  for  the  spontaneous  reduc- 
tion which  takes  place  in  this  mixture  was  automatically  adjusted  in  the 
blanks  and  standardization  by  means  of  pure  d-glucose. 


40  ct 


FIG.  1. 


EXPERIMENTAL  METHODS.  33 

THE  PROCESS  OF  REDUCTION. 

The  solution  containing  the  copper  sulphate  (solution  A)  was  run  out 
from  a  burette  of  25  c.  c.  capacity  graduated  to  0.05  c.  c.  The  alkaline 
tartrate  solution,  which  need  not  be  so  accurately  measured,  was  taken  from 
a  graduated  pipette,  and  in  the  same  amount  as  the  copper  solution.  These 
solutions  were  placed  directly  into  the  specially  designed  tube.  This  con- 
sists essentially  of  any  ordinary  centrifuge  or  sedimentation  tube  with  a 
narrow  neck,  the  volume  accurately  graduated  thereon,  and  fitted  with  a 
ground-glass  stopper.1  The  quantities  are  so  arranged  that  there  is  always 
an  ample  excess  of  copper  above  that  required  to  completely  oxidize  the 
sugar  in  the  solution  to  be  determined.  As  the  method  permits  of  such 
rapid  work,  a  rough  preliminary  estimation  can  be  made  very  quickly,  if 
necessary,  although  after  some  experience  this  is  rarely  necessary.  After 
the  introduction  of  the  copper  and  alkaline  tartrate  solutions,  the  sugar 
solution  is  run  in.  A  burette  of  25  c.  c.  graduated  to  0.05  c.  c.  was  used  for 
most  determinations.  The  contents  of  the  tube  were  then  thoroughly  mixed 
by  shaking.  It  was  found  that  for  work  with  very  small  quantities  of  sugar 
burettes  of  3  c.  c.  capacity  and  graduated  to  0.01  c.  c.  could  be  used  with 
good  results.  However,  in  the  work  with  the  cacti  there  was  always 
sufficient  material,  so  that  it  was  not  necessary  to  resort  to  this  apparatus. 

The  length  of  time  prescribed  by  various  methods  and  workers  for  heat- 
ing the  reduction  mixture  shows  a  great  variation.  The  question  has  been 
carefully  investigated  by  Peters.  From  this  work  it  becomes  clear  that 
there  is  no  advantage  in  protracted  heating  of  the  mixture ;  in  fact,  certain 
new  errors  are  thus  introduced  which  would  quite  counteract  any  advantage 
that  might  be  gained  from  such  a  procedure.  As  the  chemical  reaction  is 
actually  probably  quite  complete  only  after  long-continued  heating,*  accu- 
rate termination  of  the  process,  characterized  by  constant  values,  can  be 
attained  only  by  very  definite  and  exact  standardization  of  the  procedure. 
Thus,  as  Peters  says: 

"  It  matters  but  little  whether  the  amount  of  the  reduction  is  always  99  per 
cent  or  97.5  per  cent  of  x  if  only  the  conditions  are  so  sharply  defined  that  the 
amount  of  reduction  obtained  at  a  selected  point  is  characterized  by  great 
constancy." 

This  is  a  principle  which  is  very  generally  overlooked  in  work  with 
Fehling's  solution.  In  the  method  here  used  the  tube  is  immersed  in  a 
vigorously  boiling  water-bath  and  allowed  to  remain  therein  for  4f  minutes 
with  the  40  c,  c.  tubes  and  4  minutes  with  the  10  c.  c.  tubes.  It  was  found 
experimentally  that  at  the  end  of  this  time  practically  all  of  the  easily 
oxidizable  material  in  the  sugar  mixtures  from  the  cactus  had  been  ex- 
hausted. Almost  all  sugar  solutions  obtained  from  plant  material,  such  as 
the  ones  under  consideration,  contain  substances  other  than  sugars  which 

1  These  tubes  were  made  by  Mr.  Paul  Anders,  glass-blower  of  the  department  of 

chemistry  of  the  University  of  Illinois,  Urbana,  Illinois. 
3  NEF,  J.  U.    Liebig's  Ann.  d.  Chem.,  357,  218,  1907. 


34  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

on  prolonged  heating  may  thus  introduce  a  not  inconsiderable  error.  By 
aid  of  the  curves  of  the  rate  of  reduction  of  copper  solutions  elaborated  by 
Peters,  it  can  be  established  that  the  time  decided  upon  for  these  determi- 
nations makes  for  greatest  accuracy.  As  there  is  always  an  excess  of  copper 
in  the  solution,  there  is  danger  of  effects  inherent  to  the  use  of  too  dilute 
solutions ;  this  can  futhermore  be  easily  regulated  by  properly  preparing  the 
sugar  solution. 

At  the  expiration  of  the  requisite  time  the  tube  is  at  once  removed  from 
the  boiling  water-bath  and  rapidly  cooled  in  cold  water.  In  order  to  avoid 
cracking  of  the  tubes  it  was  found  advisable,  first,  to  wet  the  hot  tube  with  a 
little  cold  tap-water  with  the  hand  for  a  moment,  rather  than  plunge  the 
tube  into  a  bath  of  cold  water.  Thereafter  the  tube  can  be  immersed  safely. 
It  is  desirable  that  the  cooling  be  done  as  rapidly  as  possible  in  order  to 
avoid  any  resolution  of  cuprous  oxide.  The  tube  (the  contents  of  which  have 
been  cooled  to  15°)  is  then  filled  up  to  the  graduation  mark  with  distilled 
water  which  has  been  recently  boiled  in  order  to  expel  dissolved  air,  and 
the  solution  is  thoroughly  mixed,  the  tube  being  closed  with  a  ground-glass 
stopper.  The  tubes  are  then  placed  in  a  centrifugal  machine  and  revolved 
for  5  minutes.  In  the  arrangement  used  the  centrifugal  head  was  fastened 
to  the  chuck  of  an  electric  motor  of  1,700  r.  p.  m.  After  centrifuging  for 
5  minutes,  the  cuprous  oxide,  together  with  any  other  solid  or  suspended 
matter  that  may  have  been  in  the  sugar  solution,  is  thrown  down  in  a  com- 
pact mass  in  the  bottom  of  the  tube.  Occasionally  when  a  sugar  solution  was 
used  containing  considerable  foreign  matter,  a  small  amount  of  cuprous 
oxide  would  settle  on  the  sides  of  the  tube.  By  shaking  a  little  and  centri- 
fuging again  this  was  readily  clarified. 

The  obvious  advantage  of  this  procedure  is  that  the  cuprous  oxide  is  in  a 
small  compact  mass  removed  from  access  to  the  air,  and  the  danger  of 
resolution  is  thus  avoided.  A  perfectly  clear  solution  of  the  residual  copper 
solution  is  obtained  without  the  employment  of  any  filter  or  transference  to 
another  vessel.  The  entire  operation  can  be  carried  out  in  a  very  short  time, 
and  it  remains  then  simply  to  determine  the  amount  of  unchanged  copper 
left  in  solution.  Sugar  values  derived  from  these  data  are  based  upon  a 
standardization  of  the  copper  solutions  by  means  of  pure  d-glucose,  accord- 
ing to  the  data  of  Peters. 

THE  DETERMINATION  OF  COPPER. 

Peters1  has  worked  out  the  conditions  essential  for  obtaining  accurate 
results  of  copper  determination  by  means  of  the  iodide  method.  The  essen- 
tial features  of  these  findings  have  been  applied  to  the  present  circum- 
stances. Aliquot  portions  of  the  clear  supernatant  liquid  in  the  tubes  after 
centrifuging  are  removed,  by  means  of  a  pipette,  into  150  c.  c.  Erlenmeyer 
flasks.  These  are  acidified  with  concentrated  sulphuric  acid.  It  is  necessary 

*  PETEBS,  A.  W.  The  sources  of  error  and  the  electrolytic  standardization  of  the  con- 
ditions of  the  iodide  method  of  copper  analyses.  Jour.  Amer.  Chem.  Soc., 
34,  422-454,  1912. 


EXPERIMENTAL  METHODS.  35 

to  have  an  excess  of  acid  in  order  to  obtain  complete  evolution  of  iodine, 
about  1  c.  c.  of  the  concentrated  acid  for  each  10  c.  c.  of  Fehling's  solution. 
Where  a  copper  solution  other  than  Fehling's  solution  is  used,  the  amount  of 
acid  added  is  naturally  regulated  to  the  concentration  of  the  alkali.  Too 
great  an  excess  of  acid  is  also  to  be  avoided,  as  under  such  conditions  iodine 
is  liberated  from  potassium  iodide  in  the  presence  of  air.  The  flasks  are 
then  cooled  to  a  temperature  of  not  below  15°  nor  above  20°.  This  is  very 
important.  After  acidification  and  cooling,  a  solution  of  potassium  iodide 
is  added  to  each  flask. 

The  reaction  between  copper  sulphate  and  potassium  iodide  runs  to  com- 
pleteness only  when  there  is  present  a  considerable  excess  of  the  iodide; 
hence  it  is  essential  that  the  iodide  be  high  in  comparison  with  the  equivalent 
of  copper  present  and  also  in  relation  to  the  final  volume  at  the  end  of  titra- 
tion.  A  saturated  solution  is  therefore  used,  and  for  each  cubic  centimeter  of 
the  original  copper  solution  remaining,  about  0.5  c.  c.  of  the  iodide  solution 
is  used.  A  solution  of  sodium  iodide  may  also  be  used.  The  final  volume  of 
the  titration  mixture  was  usually  30  to  40  c.  c. 

The  iodine  which  is  evolved  is  titrated  immediately  with  sodium  thio- 
sulphate,  using  a  fresh  solution  of  soluble  starch  (Merck)  as  indicator, 
which  is  added  near  the  end  of  the  titration.  The  concentration  of  the 
thiosulphate  used  can  be  adjusted  according  to  the  amount  of  copper  to  be 

determined,  thus  assuring  the  greatest  accuracy  under  different  conditions. 

N-  -\r 

The  concentrations  of  the  thiosulphate  generally  used  were  —  and  — .    It 

is,  of  course,  essential  that  the  thiosulphate  produce  a  definite  end-point  in 
the  titration. 

The  presence  of  the  slightly  yellowish  cuprous  iodide  makes  the  recog- 
nition of  the  end-point  a  little  more  difficult  than  in  a  simple  iodine  titration. 
Toward  the  end  of  the  titration  the  mixture  assumes  a  very  light  brown  or 
lavender  color,  which  changes  to  a  cream  or  very  faint  pink  when  the  last 
drop  is  added.  The  end-point  can  be  verified  best  by  means  of  back  titration 
with  an  iodine  solution  of  the  same  concentration  as  the  thiosulphate,  which 
is  a  good  precaution  for  very  accurate  work.  Where  there  are  a  number  of 
determinations  to  be  made  the  various  steps  of  measuring  out  the  solutions> 
heating,  centrifuging,  cooling  the  copper  solutions,  and  titrating  can  be  so 
arranged  that  the  operator  is  constantly  busy  and  does  not  have  to  wait.  An 
interval-timer  with  alarm  attachment  was  found  very  helpful. 

THE  STANDARDIZATION  OF  THE  SOLUTIONS. 

The  copper  value  of  the  thiosulphate  solution  is  first  determined  by  going 
through  the  entire  procedure,  heating,  centrifuging,  etc.,  without  the  addi- 
tion of  any  sugar  solution.  In  this  way  any  self -reduction  of  the  alkaline 
copper  solution  is  also  adjusted.  The  dilute  solutions  of  sodium  thiosulphate 
change  in  strength  with  time,  but  a  stock  solution  of  normal  concentration 
changes  only  very  slowly.  The  sugar  value  of  the  copper  solution  is  deter- 
mined by  using  a  standard  dextrose  solution.  The  purest  material  was  used 


Sugar 
present. 


0.0012 
0.0009 
0.0125 
0.0120 
0.0122 
0.0299 
0.0287 
0.0481 
0.0484 


Sugar 
found. 


0.0012 
0.0010 
0.0125 
0.0121 
0.0122 
0.0291 
0.0290 
0.0481 
0.0486 


36  THE   CARBOHYDRATE  ECONOMY  OF   CACTI. 

for  this,  the  dextrose  supplied  by  the  United  States  Bureau  of  Standards. 
The  thiosulphate  difference  between  the  original  standardized  copper  solu- 
tion and  the  value  found  for  the  residual  copper  solution  of  a  determination 
with  sugar  represents  reduced  copper  from  which  can  be  calculated  the 
TABLE  4.  amount  of  sugar  present.  With  some  foresight,  the  sugar 
solutions  to  be  determined  can  be  so  made  up  and  the 
amount  of  copper  solution  as  well  as  the  concentration  of 
the  thiosulphate  solution  used  can  be  so  arranged  as  to  fall 
within  the  range  of  the  most  accurate  work.  This  can  be 
determined  by  reference  to.  the  valuable  table  of  Peters. 

Experiments  with  a  known  sugar  solution  gave  the  re- 
sults shown  in  table  4. 

For  very  small  quantities  of  sugar  a  more  dilute  copper 
solution  can  be  used,  though  in  this  investigation  the  above 
copper  solution  was  employed  throughout.  Thus  a  solu- 
tion composed  of  17.3  grams  copper  sulphate,  173  grams  sodium  citrate, 
100  grams  anhydrous  sodium  carbonate  made  up  to  1,000  c.  c.  with  water 
gave  good  results  with  small  quantities  of  sugar.1 

THE  ESTIMATION  OF  THE  PENTOSE  SUGARS. 

The  accurate  estimation  of  pentoses  in  mixtures  with  other  sugars  is 
associated  with  many  difficulties.  It  may,  therefore,  not  be  amiss  to  give 
here  briefly  my  experiences  and  conclusions  regarding  the  methods  of 
determination,  as  a  great  deal  of  time  was  spent  in  casting  about  for  the 
proper  means  of  estimation  of  this  group  of  sugars.  The  method  which 
has  found  most  general  application  is  that  of  Tollens,  or  one  of  the  many 
slight  modifications  thereof.  This  method  is  based  upon  the  property  of  the 
pentoses  of  their  partial  conversion  into  furfural  by  the  action  of  mineral 
acids.  The  sugar  mixture  is  distilled  with  hydrochloric  acid  (usually  12 
per  cent)  and  the  furfural  is  collected  in  the  distillate.  After  neutraliza- 
tion, the  furfural  can  be  determined  in  a  number  of  ways;  for  instance, 
the  gravimetric  estimation  of  the  phenylhydrazine  or  phloroglucine  com- 
pounds; the  titration  with  phenylhydrazine  (aniline  acetate  as  indicator) 
or  with  Fehling's  solution ;  or  the  employment  of  an  excess  of  phenylhydra- 
zine and  gasometric  determination  thereof ;  finally,  the  titrimetric  method, 
in  which  an  excess  of  sodium  bisulphite  is  added  to  form  the  aldehyde  addi- 
tion compound  and  the  unchanged  bisulphite  titrated  with  potassium  per- 
manganate.* Careful  examination  has  shown  that  all  of  these  methods  are 

1  BENEDICT,  S.  R.    A  reagent  for  the  detection  of  sugars.    Jour.  Biol.  Chem.,  5,  485- 

487,  1909. 
*  JOLLES,  A.    Ueber  ein  neues  Verfahren  zu  quantltativen  Bestimmung  der  Pentosen. 

Zeit  f.  Anal.  Chem.,  45,  196-204,  1906. 

MAQUENNE,  L.    Les  sucres  et  principaux  derives.    Page  312,  1900.    Paris. 
TOIXENS,  H.     In  Abderhalden's  Biochemische  Arbeitsmethoden,  II,  p.  99,  1910. 
Dox,  A.  W.,  and  G.  P.  PLAISANCE.    A  comparison  of  barbituric  acid,  thiobarbituric 
acid,  and  malonyguanidine  as  quantitative  precipitants  for  furfural.    Jour. 
Amer.  Chem.  Soc.,  38,  2156-2164,  1916. 


EXPERIMENTAL  METHODS.  37 

open  to  serious  error  and  are  not  reliable  in  a  quantitative  sense.  Of  special 
importance  is  the  fact  that  12  per  cent  hydrochloric  acid  is  not  without 
effect  upon  the  furfural  formed  from  the  sugars.  The  amount  of  furfural 
also  varies  with  the  proportion  of  pentose  in  the  original  mixture;  it  is 
necessary  to  apply  a  special  coefficient  for  all  concentrations.  In  using  the 
gravimetric  method  corrections,  not  altogether  satisfactory,  are  to  be 
applied  for  the  solubility  of  the  condensation  product;  on  account  of  the 
instability  of  solutions  of  phenylhydrazine,  this  method  is  also  associated 
with  great  inconvenience.  Jolles's  method  of  titration  with  sodium  bisul- 
phite has  many  advantages.  However,  as  the  proportion  of  pentose  is  some- 
times very  small  and  the  furfural  solution  therefore  exceedingly  dilute,  the 
method  is  by  no  means  reliable.  Other  and  perhaps  better  substances  for  the 
precipitation  of  furfural  have  been  suggested ;  however,  these  do  not  solve 
the  main  difficulty. 

It  must  also  be  borne  in  mind  that  pentoses  are  by  no  means  the  only 
substances  that  yield  furfural  on  treatment  with  mineral  acids.  The 
hexoses,  cane-sugar,  and  many  other  substances  found  in  plants  also  form 
furfural  under  these  conditions.  Davis  and  Sawyer1  have  shown  that  a 
mixture  of  0.01  gram  of  arabinose  plus  25  grams  cane-sugar  yielded  20  per 
cent  more  furfural  than  without  the  cane-sugar,  while  0.02  gram  arabinose 
plus  0.25  gram  cane-sugar  (the  proportion  usually  present  in  plant  extracts 
with  which  they  were  working)  was  15  per  cent  high  for  pentose  alone. 
Besides  the  hexose  and  disaccharides,  furfural  is  also  formed  from  starch, 
cellulose,  and  oxycellulose,  as  well  as  glucuronic  acid.  Furthermore,  rham- 
nose  and  the  methyl  pentoses  by  the  treatment  with  hydrochloric  acid  yield 
methyl-furfural,  which  by  the  usual  methods  can  be  easily  mistaken  for 
furfural. 

In  view  of  these  facts  it  appears  that  the  only  reliable  method  of  esti- 
mating pentoses  must  be  based  upon  a  separation  of  these  sugars  from  other 
substances.  At  present  the  most  practical  way  of  doing  this  is  by  means  of 
fermenting  away  the  hexoses.  A  good  culture  of  baker's  yeast  serves  this 
purpose  admirably,  followed  then  by  the  procedure  with  alkaline  copper 
solution  already  described.  Experimental  mixtures  of  1-arabinose  or 
1-xylose  with  dextrose  and  cane-sugar  showed  that  the  latter  were  entirely 
removed,  while  the  pentoses  remained  unaffected.  The  removal  by  distilla- 
tion of  the  alcohol  and  other  products  formed  in  the  fermentation  is 
naturally  essential.  It  is,  of  course,  necessary  to  make  certain  that  the 
non-fermentable  residue  is  actually  pentose  and  not  some  other  non-ferment- 
able sugar — for  instance  the  interesting  d-mannoketo-heptose  described  by 
La  Forge,2  which  is  occasionally  present  in  plants. 

1  DAVIS,  W.  A.,  and  G.  C.  SAWYEB.  The  estimation  of  carbohydrates.  The  presence 
of  free  pentoses  in  plant  extracts  and  the  influence  of  other  sugars  on  their 
estimation.  Jour.  Agr.  Set.,  6,  406-412,  1915. 

*  LA  FOBGE,  F.  B.  D-mannoketoheptose,  a  new  sugar  from  the  avocado.  Jour.  Biol. 
Chem.,  28,  511-522,  1917. 


38  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

C02  DETERMINATION. 

The  method  employed  for  determining  the  rate  of  CO2  emission  was 
based  upon  the  absorption  of  the  carbon  dioxid  in  a  standard  solution  of 
barium  hydroxide  and  titration  of  the  unchanged  base.  The  spines  were 
first  carefully  removed  from  the  cactus  joints,  weighed,  and  the  cut  ends 
were  covered  with  a  soft  wax  preparation  to  prevent  a  traumatic  effect. 
The  joints  (usually  4  to  6  were  employed)  were  placed  in  a  desiccator  pro- 
vided with  entrance  and  exit  tubes.  The  desiccator  was  then  hermetically 
sealed,  and  a  light-proof  cover  placed  thereover.  This  was  immersed  com- 
pletely in  a  Freas  electric  water  thermostat  and  the  tubes  were  connected. 
The  air  was  drawn  through  the  apparatus  by  means  of  a  specially  devised 
electric  pump  and  the  pressure  was  regulated  by  a  Palladin  pressure 
regulator.  The  air  passed  first  through  a  train  of  moist  soda-lime,  then 
through  a  25-foot  coil  of  glass  tubing  in  the  thermostat,  into  the  respiration 
chamber ;  from  there  through  a  water-trap,  and  finally  through  the  Meyer's 
tubes  containing  125  c.  c.  of  0.1  normal  barium  hydroxide.  A  series  of 
tubes  was  so  arranged  that  the  stream  of  air  could  be  passed  into  a  fresh 
tube  without  interrupting  the  experiment.  A  single  tube  collected  the 
carbon  dioxid  for  from  4  to  12  hours,  and  the  experiment  was  allowed  to 
run  usually  from  48  to  60  hours.  The  barium  hydroxide  together  with  the 
precipitated  barium  carbonate  was  poured  into  a  narrow  bottle,  sealed,  and 
the  carbonate  allowed  to  settle.  After  24  hours  aliquot  portions  of  the  clear 
solution  were  drawn  off  with  a  pipette  and  titrated  with  standard  hydro- 
chloric acid,  using  methyl  orange  as  indicator.  The  amount  of  carbon 
dioxid  per  unit  time  can  be  easily  calculated  from  the  difference  in  strength 
of  the  barium  hydroxide  solution  between  the  original  and  after  the  air 
from  the  plant  had  passed  through. 


THE  CARBOHYDRATES  OF  THE  CACTI. 


TABLE  5. 


Water  

P.  et. 
95  00 

p.  et. 
75  00 

Crude  protein.. 

0  50 

1  00 

Carbohyrdrates  hydroly- 
zable  with  1  per  cent. 
HC1 

2  00 

10  00 

Cellulose  

1.00 

3  00 

Crude  fat  

0  25 

0  50 

Ash  

1  00 

3  50 

TABLE  6. 


IV.  THE  CARBOHYDRATES  OF  THE  CACTI. 

As  has  already  been  indicated,  the  cacti  consist  essentially  of  carbo- 
hydrate material.  Compared  to  this  the  protein  and  fats  are  present  in  only 
small  quantities,  nor  does  the  content  in  the  plant  of  these  latter  substances 

vary  appreciably  by  the  change  of  con- 
ditions which  so  decidedly  affect  the 
carbohydrates.  Roughly,  the  fresh  ma- 
terial of  the  growing  and  mature  joints 
is  composed  about  as  shown  in  table  5. 
Griffiths  and  Hare1  give  the  fol- 
lowing analysis  (table  6)  of  the  ash 
of  Opuntia  phoeacantha  collected  in  the 
Santa  Rita  Mountains  near  Tucson, 
Arizona. 

Individual  plants  show  a  considerable  variation  in  all  their  components, 
depending  upon  their  location  and  the  general  complex  of  environmental 
conditions. 

The  differences  in  carbohydrate-content  and 
in  the  proportion  of  the  various  sugars  be- 
tween plants  growing  in  the  desert  at  Tucson 
and  in  the  cool,  humid  climate  of  Carmel, 
California,  is  illustrated  in  the  accompany- 
ing analyses  of  Opuntia  phceacantha  during 
September  (table  8).  The  values  are  in  per 
cent  of  the  fresh  weight  An  analysis  of  the  causes  which  induce  these 
differences  in  composition  will  be  taken  up  later. 

For  comparison  analyses  are  given 
in  table  8  of  Opuntia  phoeacantha 
and  of  Opuntia  versicolor,  the  latter 
being  relatively  free  of  the  mucilagi- 
nous substances  so  profuse  in  the 
former. 

It  is  evident  that  the  major  por- 
tion of  the  carbohydrates  in  these 
plants  is  in  the  form  of  polysaccha- 
rides  and,  as  will  be  seen,  the  simpler 


Fe 

0  15 

Na 

0  25 

Al  .  .  .  . 
Mn.... 
Ca  
Mg.... 

.  0.15 
.  0.37 
.29.00 
.   6.06 
.  6.40 

SiO,  . 
PO4.. 
So4  .. 
01... 
CO, 

...  0.91 
...  2.64 
...  0.82 
...  0.55 
45  97 

TABLE  7. 


Carmel. 

Tucson. 

Water 

91.15 
2.61 
1.94 
0.09 
0.07 
0.52 
0.14 
1.70 

80.34 
4.30 
3.50 
1.65 
0.04 
0.06 
0.05 
1.74 

Total  polysaccharides  
Hexose  polysaccharides  .  .  . 
Disaccharides  

Hexoses    

Pentoses    ...                 ... 

Pentosans 

sugars  (monosaccharides  and  disaccharides)  vary  greatly  with  condi- 
tions, both  in  total  content  and  in  the  proportion  to  the  polysaccharides. 
A  separation  of  the  monosaccharides  and  disaccharides  from  the  polysac- 
charides was  obtained  by  means  of  thorough  extraction  with  alcohol,  the 
first  two  groups  going  completely  into  alcohol,  while  the  latter  are  insoluble. 
The  acid  hydrolysis  portion  contained  the  hydrolyzed  polysaccharides  as 
well  as  the  monosaccharides  and  disaccharides. 


GRIFFITHS,  D.,  and  R.  P.  HARE.    Prickly  pear  and  other  cacti  as  food  for  stock.    Bull. 
New  Mexico  Agri.  Expt.  Station,  60,  15,  1906. 


40 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


The  polysaccharides  are  present  mainly  in  the  form  of  starch  and  also 
as  a  mucilaginous  substance  of  pentosan  nature.  As  was  explained  in  the 
section  on  methods  of  analysis,  it  was  unfortunately  impossible  to  determine 
the  starch  quantitatively.  However,  by  means  of  the  iodine  reaction  it 
became  evident  that  the  starch  is  present  in  the  form  of  very  numerous 
small  starch  grains.  The  Schimper  chloral  hydrate-iodine  reagent  proved 
the  most  satisfactory  for  the  detection  of  these.  From  the  variation  in  size 
and  number  of  the  starch  grains  a  general  idea  could  be  gained  as  to  the 
amount  of  this  substance,  though,  of  course,  this  is  not  an  accurate  measure 
and  reveals  only  wide  differences.  It  is,  nevertheless,  a  reliable  method  for 
the  detection  of  the  presence  of  starch  and  can  be  used  (to  a  measure)  to 
indicate  the  fate  of  this  material  under  the  various  experimental  conditions 
hereinafter  discussed. 

TABLE  8. — Results  of  analyses  of  0.  versicolor  and  0.  phwacantha  in 
percentages  of  the  fresh  and  dry  material. 


Opuntia  versicolor. 

Opuntia  phaeacantha. 

Fresh. 

Dry. 

Fresh. 

Dry. 

Water  

82.15 
1.97 
1.50 
0.33 
1.59 
1.29 
0.15 
0.34 
0.18 
0.36 
0.16 
0.23 

ii.05 
8.40 
1.87 
8.92 
7.20 
0.86 
1.89 
1.01 
2.01 
0.88 
1.13 

78.70 
3.53 
3.22 
0.24 
1.81 
1.59 
0.08 
0.26 
0.16 
1.64 
0.10 
1.55 

ieleo 

15.15 
1.10 
8.50 
7.44 
0.36 
1.20 
0.74 
7.70 
0.46 
7.26 

Total  sugars  

Total  polysaccharides  
Disaccharides  and  hexoses.  . 
Total  hexose  sugars. 

Hexose  polysaccharides  

Monosaccharides  

Hexose  

Total  pentose  

Pentose  

Pentosan         .... 

The  method  adopted  for  the  hydrolysis  of  the  polysaccharides  has  already 
been  described.  In  order  to  establish  the  nature  of  the  carbohydrates  in 
this  hydrolyzed  portion,  the  following  procedure  was  carried  out:  The 
neutralized  solution  from  the  hydrolysis  was  evaporated  down  at  reduced 
pressure.  The  residue,  which  contained  a  great  deal  of  salt,  was  extracted 
with  hot  alcohol ;  this  alcohol  solution  was  evaporated  at  reduced  pressure ; 
the  residue  was  dissolved  in  a  very  little  water  and  a  large  quantity  of 
alcohol  added.  Hereby  more  salt,  as  well  as  some  gum,  was  thrown  out  of 
solution.  This  was  repeated  twice,  after  which  the  sugar  residue  from  the 
evaporation  of  the  alcohol  showed  but  a  trace  of  inorganic  ash  on  ignition. 
This  residue  contained  all  the  sugars  present  in  the  cactus,  hydrolyzed  to 
monosaccharides.  It  consisted  of  a  clear  brown  gum.  The  aqueous  solution 
was  treated  with  a  small  quantity  of  lead  acetate ;  the  insoluble  portion  was 
filtered  off;  the  lead  in  solution  was  precipitated  with  a  slight  excess  of 
sulphuric  acid,  and  the  lead  sulphate  was  filtered  off.  The  sulphuric  acid 
was  then  carefully  removed  by  precipitation  with  barium  hydroxide.  After 


THE  CARBOHYDRATES  OF  THE  CACTI.  41 

this  treatment  the  sugar  solution  was  somewhat  lighter  in  color.  It  was 
subsequently  treated  with  blood  charcoal,  which  produced  a  light-yellow 
solution.  From  200  grams  of  the  dry  cactus  material  25.25  grams  sugar 
gum  were  thus  obtained.  The  solution  was  neutral  towards  litmus,  reduced 
Fehling's  solution  strongly,  and  was  dextro-rotatory,  [a]^  =  -(-25.60°. 

The  mixture  was  tested  for  the  presence  of  galactose  in  the  usual  manner 
by  treatment  with  nitric  acid,  sp.  g.  1.15.  No  mucic  acid,  however,  was 
obtained,  which  indicated  the  absence  of  galactose.  The  acid  solution  thus 
obtained  from  the  oxidation  with  nitric  acid  was  made  alkaline  with 
potassium  carbonate  and  then  treated  with  an  excess  of  acetic  acid.  On 
standing,  crystals  of  mono-potassium-saccharate  separated  out;  this  indi- 
cated the  presence  of  glucose  in  the  sugar  mixture.  The  presence  of  fructose 
was  established  by  means  of  the  resorcin  reaction. 

A  portion  of  the  sugar  mixture  was  fermented  with  pure  baker's  yeast, 
as  has  already  been  described.  The  residue  from  this  process  was  a  light- 
yellow  gum.  In  solution  this  evolved  furfural  freely  when  heated  with 
12  per  cent  sulphuric  acid,  and  gave  an  intense  violet-red  coloration  with 
phlorogluzin.  The  reactions  indicate  the  presence  of  a  pentose  sugar.  The 
specific  rotation  in  a  1  dm.  tube  was  [a]*f  =  -{-22.1°  with  [a]  =  -f2.05° 
and  9.26  grams  in  100  c.  c.  of  water.1  The  specific  rotation  of  the  solution 
did  not  change  after  standing  24  hours. 

A  portion  of  the  sugar  was  treated  with  2  parts  of  phenylhydrazine 
hydrochloride  and  3  parts  of  crystalline  sodium  acetate  and  heated  for  an 
hour  on  the  boiling  water-bath.  On  cooling,  a  large  quantity  of  bright 
yellow  phenylosazon  separated  out.  This  was  recrystallized  from  water 
and  again  from  methyl  alcohol.  After  drying  in  vacuum  over  sulphuric 
acid,  this  melted  with  gas  evolution  at  159°.* 

Another  portion  of  the  sugar  mixture  was  treated  in  aqueous  solution 
with  an  excess  of  bromine  and  powdered  cadmium  carbonate.  After  24 
hours  the  excess  of  bromine  was  driven  off  by  heating  on  the  water-bath, 
the  mixture  was  filtered  from  unchanged  CdCO3,  and  tie  filtrate  was 
evaporated  almost  to  dryness,  and  then  2  c.  c.  of  alcohol  was  added  thereto. 
After  several  days  the  characteristic  crystals  separated  out  of  the  double  salt 
cadmium  bromide  xylonate,* 

Cd(C6H906)2CdBr2  -  2H20  •     !>]«•=  +6.9°. 

Prom  these  results  it  is  evident  that  the  pentose  sugar  was  1-xylose. 

In  the  alcoholic  extract  of  the  dry  plant  material  there  would  be  present 
of  the  carbohydrates  only  the  disaccharides  and  monosaccharides.  The 
extract  was  prepared  as  has  been  previously  described.  The  alcohol  was 
distilled  off  at  reduced  pressure.  The  residue  contained,  besides  the  sugars, 
a  large  amount  of  chlorophyll  and  oil.  To  this  was  added  water  and  some 

1  MAQUENNE,  L».    Les  sucres  et  principaux  derives,  p.  350.        *  Ibid.,  p.  356. 

'  BEBTBAND,  M.  G.    Recherches  sur  quelques  derives  du  xylose.    Bull.  Soc.  Chlm.,  m, 

5,  546,  554,  1891. 
NEF,  J.  U.    Ann.  d.  Chem.,  403,  253,  1»13. 


42  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

blood  charcoal  and  the  mixture  was  heated  on  the  water-bath.  After  such 
treatment  the  solution  could  be  easily  filtered  and  the  filtrate  was  light 
brown  in  color.  As  has  been  described,  the  disaccharides  in  this  mixture 
were  determined  by  hydrolysis  with  hydrochloric  acid  and  the  determina- 
tion of  the  cupric  reducing  value.  The  amount  of  maltose  was  exceedingly 
small,  as  was  indicated  by  the  fact  that  the  difference  was  very  little  between 
solutions  hydrolyzed  with  hydrochloric  acid  and  with  an  invertase  prepara- 
tion. Equal  amounts  of  the  same  sugar  solution  were  treated,  the  one  with 
hydrochloric  acid  in  the  manner  already  described,  the  other  with  a  solution 
of  invertase.1  Thus  100  c.  c.  of  the  solution  hydrolyzed  with  acid  required 
22.20  c.  c.  copper  solution,  while  the  same  amount  treated  with  invertase 
took  23.30  c.  c. 

The  presence  of  glucose  and  fructose  in  the  alcoholic  extract  was  also 
established,  as  in  the  case  of  the  hydrolyzed  portion.  The  mixture  was 
fermented  with  baker's  yeast  in  the  usual  manner  in  order  to  determine  the 
nature  of  the  pentose  sugars.  By  treatment  with  phenylhydrazine-hydro- 
chloride  and  sodium  acetate,  phenylxylosozone  was  obtained,  which  was 
purified  by  recrystallization  from  methyl  alcohol.  The  specific  rotation  of 
the  pure  gum  was  found  [a] *°  =-{-19.0°  to  -j-21.00  from  several  prepa- 
rations. Here,  however,  a  difficulty  was  encountered.  After  the  aqueous 
solution  of  the  gum  had  stood  for  24  hours  it  no  longer  showed  the  same 
specific  rotation,  but  had  dropped  considerably,  and  in  a  few  cases  had  even 
become  levo-rotatory.  In  the  cause  of  this  phenomenon  lay  the  substantia- 
tion of  the  theory  of  pentose  formation  which  had  been  previously  formu- 
lated and  which  is  discussed  in  the  section  on  the  pentose  sugars.  This 
reduction  of  the  specific  rotation  (as  was  finally  established)  was  caused  by 
glucuronic  acid.  The  specific  rotation  of  1-xylose  is  [a]*,0  =  +20°,  that  of 
glucuronic  lactone  is  given  by  Fischer  as  [a]!)0^  -f-  19. 10.1 

A  portion  of  the  gum  which  showed  this  property  of  decreasing  the 
specific  rotation  on  standing  in  water  solution  was  thoroughly  dried  by 
evaporating  the  water  at  reduced  pressure  from  a  boiling  water-bath.  The 
flask  containing  the  gum  was  allowed  to  remain  open  for  several  months 
during  the  hot,  dry  summer.  The  gum  was  then  dissolved  in  hot  water  and 
treated  for  5  minutes  with  blood  charcoal.  After  filtering,  the  water  was 
again  distilled  off  at  reduced  pressure.  During  this  process  small  pure- 
white  crystals  separated  out  from  the  solution,  and  considerably  more  of  the 
same  substance  crystallized  out  on  cooling.  This  was  filtered  off  and  dried 
in  vacuum  over  sulphuric  acid.  The  substance  showed  the  following 
properties :  in  water  solution  it  reduced  Fehling's  solution  strongly ;  when 
heated  with  12  per  cent  sulphuric  acid,  furfural  was  liberated  freely;  it 
gave  the  red  color  with  2  per  cent  phloroglucine  and  hydrochloric  acid; 

1  For  this  purpose  the  best  invertase  preparation  was  obtained  by  the  process 
described  by  W.  A.  Davis,  in  The  use  of  enzymes  and  special  yeasts  in  carbo- 
hydrate analysis.  Jour.  Soc.  Chem.  Ind.,  35,  201-240,  1916. 

'  FISCHEB,  E.,  and  O.  PUXXTY.  Reduction  der  Zuckersaeure.  Ber.  d.  deut  chem.  Ges., 
24,  523,  1891. 


THE  CARBOHYDRATES  OF  THE  CACTI.  43 

10  c.  c.  of  the  solution  with  1  to  2  drops  of  15  per  cent  alphanaphthol  and 
3  c.  c.  concentrated  sulphuric  acid  (Goldschmiedt  reaction)  gave  the  beau- 
tiful emerald  green  coloration  characteristic  of  glucuronic  acid.  Special 
experiments  with  a  large  variety  of  substances  apt  to  be  found  in  plants 
showed  that  none  of  these  except  nitrates  produce  the  green  color.  The 
absence  of  nitrates  was  established  by  the  diphenylamine  reaction.  The  hot 
aqueous  solution  was  distinctly  acid. 

The  substance  under  discussion  melted  sharply  at  176°  and  burned  with- 
out leaving  any  ash.  The  specific  rotation  was  [^J^  =  +20.0°  ;  that  is, 
1.25  grams  in  100  c.  c.  in  a  1  dm.  tube  gave  [a]  =  -{-0.25°.  These  prop- 
erties establish  the  identity  of  this  substance  with  glucuronic  acid.  After 
28  hours  the  same  solution  showed  a  specific  rotation  of  [<*]™  =  — 3.2°. 

It  is  a  well-known  property  of  the  type  of  acid  to  which  glucuronic  acid 
belongs,  that  the  lactones  of  the  acids  are  usually  strongly  rotatory,  but  the 
free  acids  are  only  feebly  so.  Nef 1  has  worked  out  the  conditions  of  equi- 
librium for  d-mannonic  acid.  The  normal  y3-lactone  of  d-mannonic  acid  in 
water  solution  has  a  specific  rotation  of  -{- 111.1°.  On  standing  at  20° 
this  solution  changes,  so  that  after  24  hours  about  75  per  cent  of  the  lactone 
is  converted  into  free  d-mannonic  acid  and  the  rotation  has  dropped  to 
[a]  |o  =-|-280.  The  free  d-mannonic  acid  has  a  rotation  of  [a]*,0  = 
— 1.0°.  From  the  data  given  it  appears  that  glucuronic  acid  behaves  in 
a  similar  manner  and  the  reduction  of  the  rotation  is  to  be  ascribed  to  the 
conversion  of  a  portion  of  the  dextro-rotatory  lactone  into  the  feebly  levo- 
rotatory  free  acid. 

This  is  the  first  time  that  glucuronio  acid  has  been  reported  as  a  plant 
constituent/  It  is,  however,  probably  a  rather  common  component  of 
plants,  though  its  isolation  is  associated  with  considerable  diificulty.  Re- 
peated attempts  with  the  cacti  have  yielded  only  very  small  amounts.  From 
100  grams  of  the  dry  material,  or  about  500  grams  of  the  fresh  cactus, 
0.1  gram  of  pure  glucuronic  lactone  was  obtained.  The  role  of  this  sub- 
stance in  the  carbohydrate  metabolism  will  be  discussed  in  the  section  on  the 
origin  and  role  of  pentose  sugars. 

One  of  the  most  striking  characteristics  of  the  cacti  is  their  high  mucilage 
content.  This  property  adds  enormously  to  the  difficulty  of  working  with 
them  and  necessitates  quite  a  different  procedure  of  analytical  and  experi- 
mental technique  from  that  commonly  employed  in  work  of  this  nature. 
Some  of  these  peculiarities  have  already  been  discussed  in  the  preceding 
chapter.  The  presence  of  mucilages  or  slimes  has  been  very  generally 
observed  in  succulent  plants  and  many  fleshy  vegetable  tissues,  and  is 
undoubtedly  of  great  physiological  importance.  In  fact,  in  one  form  or 
another,  these  colloidal  substances  are  present  in  all  plants  to  a  greater  or 
less  extent.  The  exact  nature  of  these  substances  has  not  been  definitely 

1  NEP,  J.  U.    Liebig's  Ann.  d.  Chem.,  403,  308,  1913. 

a  PALLADIN,  W.    Glucuronic  acid,  glucuronides  and  glyoxylic  acid  in  plants.    Chem. 
Abstracts,  11,  48,  351,  1917. 


44  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

established  as  yet,  nor  is  there  any  satisfactory  scheme  of  classification  or 
identification,  for  different  properties  have  been  emphasized  and  varying 
results  obtained  from  considering  the  morphological,  functional,  or  chemical 
aspects.  But  from  none  of  these  aspects  has  a  satisfactory  or  comprehensive 
view  been  gained  of  the  nature  or  behavior  of  the  mucilages.  Hemicellulose, 
pectin,  slime,  and  gum  are  names  which  have  no  definite  or  universal  conno- 
tation either  as  to  origin,  structure,  or  chemical  composition,  although  some 
efforts  have  been  made  for  a  system  of  classification.1  All  of  these  sub- 
stances are  carbohydrates  in  which  a  number  of  simpler  sugars  are  con- 
densed to  form  highly  complex  compounds.  It  is  very  questionable  whether 
any  of  these  have  ever  been  obtained  in  a  pure  state,  and  it  seems  very 
probable  that  in  most  cases  mixtures  of  carbohydrates  as  well  as  of  other 
substances  have  been  dealt  with.  It  has  been  known  for  some  time  that 
complex  organic  acids,  such  as  gluconic  acid  and  a  number  of  gum  acids,  are 
also  components,  though  their  exact  nature  and  the  manner  in  which  these 
are  placed  in  the  compounds  are  not  definitely  known.*  Furthermore,  a 
small  amount  of  proteins  and  other  nitrogenous  substances  as  well  as 
inorganic  salts  usually  are  present  as  admixtures.  All  of  these  substances, 
hemicellulose,  pectins,  slimes,  and  gums,  on  hydroylsis  with  dilute  acids, 
yield  pentoses  as  well  as  hexoses,  and  they  are  therefore,  in  general,  all 
pentosans. 

It  is  indeed  not  surprising  that  such  difficulty  has  been  encountered  in 
working  out  specific  reactions  for  the  detection  of  these  substances,  as  they 
are  so  very  much  alike  in  general  composition  and  undergo  few  character- 
istic reactions  that  could  be  utilized,  for  instance,  for  microchemical  detec- 
tion. Most  of  the  work  in  this  field  has  been  carried  out  through  micro- 
chemical  means,  and  many  of  the  tests  employed  for  characterizing  the 
various  mucilages  are  based  rather  upon  the  presence  of  admixtures  or  of 
differences  in  the  physical  state  than  upon  any  definite  chemical  property. 
As  a  matter  of  tradition  plant  slimes  and  gums  are  differentiated  in  that 
under  the  former  come  all  those  polysaccharides  which  swell  greatly  in 
water,  but  show  limited  dispersion,  and  can  not  be  drawn  out  in  threads, 
while  the  gums  are  completely  soluble  in  water.*  A  much  more  rational 
system  would  be  one  based  upon  the  nature  of  the  products  of  hydrolysis  and 
perhaps  especially  of  the  gum  acids. 

A  very  extensive  literature  has  accumulated  on  the  subject  of  these 
colloidal  polysaccharides,  dealing  mostly,  however,  with  the  cytological 
aspect  of  their  formation.  The  opinions  run  widely  divergent,  there  is  no 
uniformity  of  terms,  and  the  subject  in  general  is  a  most  uncoordinated  one. 
It  is  certain,  however,  that  the  various  forms  of  mucilage  are  produced  in  a 

1  TTJNMANN,  O.    Pflanzen  microchemie.    Pages  560-592,  1913.    Berlin. 
1  CZAPEK,  P.    Biochem.  d.  Pflanzen.  2d  edition,  p.  676  for  literature,  1913. 
•RUHLAND,  W.    Ueber  Arabinbildung  durch  Bakterien  und  deren  Beziehung  zum 

gummi  der  Amygdaleen.    Ber.  d.  deutsch.  Bot.  Ges.,  24,  393-401,  1906. 
FBANK,  A.  B.     Ueber  die  anatomische  Bedeutung  der  vegetablischen   Schleime. 

Jahr.  f.  wiss.  Bot,  5,  198,  1867. 


THE   CARBOHYDRATES  OF  THE   CACTI.  45 

variety  of  ways,  that  this  is  not  confined  to  the  metamorphosis  of  the  cell 
wall  or  middle  lamella,  and  is  often  formed  in  the  interior  of  the  cell.  The 
mucilages  are  undoubtedly  used  for  a  variety  of  purposes  by  the  plant.  The 
interest  here  is  confined  to  their  use  as  food  material  and  their  property 
of  water  imbibition. 

In  order  to  obtain  the  mucilage  in  as  pure  a  condition  as  possible  for 
further  study,  various  procedures  may  be  followed.  A  crude  product  can 
be  obtained  easily  by  first  cutting  up  the  plant  material  with  a  meat  chopper 
and  slowly  pressing  the  mass  through  a  muslin  cloth.  The  portion  which 
passes  through  is  a  thick,  clear  fluid.  However,  this  latter  material  always 
contains  reducing  sugars  and  a  little  starch. 

When  the  cactus  joints  are  placed  under  a  bell-jar  in  an  atmosphere  con- 
taining ether,  chloroform,  or  acetone,  after  about  24  hours  the  heavy 
mucilage  exudes  from  the  cut  surfaces  of  the  joint  After  several  days  a 
considerable  quantity  can  thus  be  collected. 

The  most  satisfactory  results  were  obtained  by  cutting  the  cactus  into 
pieces  of  about  1  c.  c.  These  were  placed  in  a  quantity  of  distilled  water 
equal  to  the  volume  of  the  pieces,  and  a  few  drops  of  formaldehyde  were 
added.  After  12  hours  the  mixture  was  poured  onto  a  cheese-cloth  filter 
and  the  liquid  was  allowed  to  drain  off.  The  pieces  of  cactus  were  again 
placed  in  an  equal  quantity  of  fresh  distilled  water  and  after  12  hours  the 
mass  was  again  filtered.  In  order  to  free  from  small  particles  of  tissue,  the 
filtrate  was  passed  through  a  very  fine  chiffon.  The  resulting  filtrate  was 
clear,  colorless,  and  of  about  the  consistency  of  fresh  egg  albumin.  It  had 
a  decided  acid  reaction  and,  on  heating,  it  browned  and  became  thin, 
probably  on  account  of  hydrolysis.  To  the  solution  of  the  mucilage  was 
added  3  times  the  volume  of  95  per  cent  alcohol.  This  caused  a  white, 
gelatinous  precipitate,  which  was  rapidly  filtered  and  washed.  The  amor- 
phous, elastic  mass  was  dried  in  vacuum  over  calcium  chloride.  Allowed 
to  remain  in  the  air,  it  became  gray  and  finally  dark  brown.  It  dissolved 
in  water  to  a  mucilage  with  a  small  quantity  of  insoluble  matter,  probably 
coagulated  proteins.  The  whole  original  precipitate  was,  therefore,  again 
dissolved  in  water,  filtered  from  the  small  amount  of  insoluble  matter, 
precipitated  again  with  alcohol,  filtered,  and  dried  in  the  same  way.  This 
material  dissolved  to  a  clear  thick  mucilage  in  water. 

The  solution  exhibited  still  a  slightly  acid  reaction.  It  showed  a  decided 
dextro-rotatory  power,  although  the  value  was  not  constant  in  all  prepara- 
tions. It  was  impossible  to  salt  out  the  mucilage  from  solution  with 
ammonium  sulphate  or  lead  acetate.  The  free  mucilage  gave  no  definite 
coloration  with  iodine  or  with  chlorzinc  iodine.  The  solution  did  not  show 
the  slightest  reduction  of  Fehling's  solution.  It  was,  however,  very  easily 
hydrolyzed ;  after  heating  about  10  minutes  with  0.5  per  cent  hydrochloric 
acid,  the  mixture  reduced  Fehling's  solution  very  rapidly.  A  preparation 
of  invertase  (made  as  described  above)  was,  however,  absolutely  without 
any  effect  after  48  hours,  nor  was  the  solution  affected  by  pure  baker's  yeast 


46 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


TABLE  9. 


The  hydrolysis  of  the  mucilage  with  1  per  cent  hydrochloric  acid,  and  the 
quantitative  determination  of  the  sugars  in  the  usual  way,  showed,  that  34.1 
per  cent  was  d-glucose  and  65.9  per  cent  was  1-xylose. 

The  mucilage  prepared  as  described  above  always  contained  a  small 
amount  of  inorganic  ash,  from  which  it  was  impossible  to  free  it  by  means 
of  resolution  and  precipitation  with  alcohol.  It  is  highly  probable  that  this 
indicates  the  presence  of  a  salt  of  an  organic  acid  of  the  nature  of  glucuronic 
acid  or  some  body  akin  to  the  gum  acids. 

As  to  the  localization  of  the  mucilage  in  the  cactus  and  the  visible  changes 
associated  with  its  formation,  just  a  few  observations  were  made.  Under 
the  microscope  in  fresh  sections  of  the  plant,  the  mucilage  is  most  clearly 
visible  in  special,  large  cells,  distributed  throughout  the  tissue.  These  cells 
are  two  or  three  times  the  size  of  the  rest  of  the  cells  and  are  filled  with 
mucilage.  When  the  fresh  sections  or  larger  pieces  of  the  cactus  are  placed 
in  distilled  water  the  mucilage  cells  swell  greatly  and  finally  burst,  allowing 
the  mucilage  to  pass  out.  By  cutting  sections  from  material  which  had  been 
previously  well  dehydrated  in  alcohol  and  then  allowing  water  or  30  per  cent 
alcohol  to  flow  onto  the  sections  on  the  microscopic  slide,  the  process  of 
swelling  and  eventual  bursting  of  the  cell  can  be  closely  followed.  It  is 

still  an  open  question  whether  all 
the  mucilage  obtained  comes  from 
the  bursting  of  the  mucilage  cells,  as 
separate  experiments  have  shown 
that  the  mucilage  is  capable  of  slowly 
passing  through  a  parchment  mem- 
brane. The  behavior  of  these  carbo- 
hydrates in  colloidal  solution  has  not 
been  definitely  established.  The  fact 
is  very  generally  disregarded  that 
many  emulsion  colloids  exhibit  a  decided  diffusion,  though  this  is,  of  course, 
much  lower  than  most  crystalloids.  Thus,  for  example,  Herzog  *  obtained 
the  accompanying  constants  (table  9),  using  a  modification  of  Graham's 
method. 

A  case  of  the  passage  of  gum  arabic  through  living  membranes  exists 
where  this  substance  is  used  to  keep  up  the  blood  volume  in  animals  and 
is  found  to  be  excreted  in  the  urine.*  In  the  case  of  the  flowing  of  the 
mucilage  from  the  anesthetized  plant  it  would  seem  that  here  the  ether, 
chloroform,  or  acetone  affect  the  membranes  and  cell  walls  so  that  there  is 
a  relatively  rapid  diffusion.  Very  recently  Lloyd  *  has  devised  methods  of 
staining  the  mucilage  cells  in  situ,  showing  in  a  beautiful  way  the  distribu- 
tion and  behavior  of  these  cells  and  their  contents. 

*  FREUNDLICH,  H.    Kapillarchemie.    Page  402,  1909.    Leipzig. 

*HOGAN,  J.  J.,  and  MARTIN  H.  FISCHEB.    Zur  Theorie  und  Praxis  der  Transfusion. 

Kolloidchem.  Beihefte,  3,  385-416,  1912. 

HTJBWITZ,  P.  H.    Intravenous  injections  of  colloidal  solutions  of  Acacia  in  hemor- 
rhage.   Jour.  Amer.  Med.  Assn.,  68,  699-701,  1917. 

1  LLOYD,  P.  E.    Year  Book,  Carnegie  Inst.  Wash.  1918,  p.  72. 


Substance. 

Temp. 

•>*£•'«•• 

Ovalbumin 

15  5° 

0  063 

7  5 

0  039 

12  0 

0  073 

16  6 

0  041 

Emulsin  

15.3 

0.042 

Urea  (M  —  60)  

18.0 

1.01 

Glucose  (M=170).... 

18.0 

0.57 

THE  CARBOHYDRATES  OF  THE  CACTI. 


47 


Of  importance  for  the  present  consideration  is  the  fact  that  the  cells  of 
the  cactus  under  normal  conditions  contain  many  minute  starch  grains. 
However,  in  these  mucilage  cells  the  starch  grains  are  either  very  few  in 
number  or  absent  entirely.  When  the  microscopic  sections  or  pieces  of 
cactus  1  c.  c.  in  size  are  washed,  and  then  placed  in  water,  the  starch  in  the 
mucilage  cells  disappears  within  18  hours.  The  starch  in  the  other  cells 
also  disappears,  though  more  slowly,  2  to  6  days,  according  to  the  material. 
During  the  process  there  is  no  loss  of  cane-sugar  or  reducing  sugar  from 
the  plant  into  the  water.  In  similar  material,  kept  so  that  the  water-content 
of  the  plant  does  not  change  greatly,  the  starch  remains  for  several  weeks, 
while,  on  the  other  hand,  in  the  pieces  of  plant  which  are  dehydrated,  as,  for 
instance,  partially  drying  in  the  air  or  over  calcium  chloride,  there  is  an 
increase  in  the  starch  in  all  cells.  These  facts  are  of  importance  in  con- 
sideration of  the  carbohydrate  equilibrium  to  be  discussed  later.  The 
immediate  source  of  formation  of  this  mucilage  has  not  been  absolutely 
established,  though  from  Lloyd's  observations  it  would  appear  that  the 
mucilage  in  some  cases  is  a  product  of  the  TABLE  10._jr/OM  6y  evaporation 
cell  wall.  This  is  important  in  connection  of  mucilage  and  water  from 
with  the  observations  of  MacDougal,  Long, 
and  Brown  on  the  formation  of  lacunae  in 
the  starved  Bisnaga  already  referred  to.  The 
theory  of  the  chemistry  of  mucilage  and 
pentosan  formation  will  be  taken  up  under 
the  chapter  on  respiration. 

Finally,  it  may  be  of  interest  to  note  some 
experiments  on  the  water-holding  power  of 
the  mucilage,  as  this  has  often  been  con- 
sidered the  chief  function  of  these  substances 
in  the  succulent  xerophytes.  The  mucilage  had  a  specific  gravity  of  1.017 
and  was  placed  in  wide-mouth  weighing-bottles,  of  as  nearly  the  same  size 
as  could  be  obtained.  Comparisons  were  made  with  tap-water.  The  bottles 
were  placed  on  a  table  1  meter  in  diameter,  which  revolved  once  every  2 
minutes.  Weighings  were  made  every  hour.  The  difference,  it  will  be 
noticed,  is  very  slight  until,  of  course,  the  evaporation  is  retarded  through 
the  formation  of  a  slight  film  on  the  mucilage. 


equal  surfaces,  in  grams. 


Time. 

Mucilage. 

Water. 

1  hour  

2.4402 
2.4157 

2.4601 
2.4187 

2  hours  

3.1068 
3.0895 

3.1900 
3.2112 

3  hours  

4.6807 
4.6653 

5.2711 
4.5691 

4  hours  .... 

5.2185 
5.3573 

5.5291 
5.5844 

THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


V.  SEASONAL  VARIATIONS  IN  THE  CARBO- 
HYDRATE-CONTENT. 

The  vegetation  of  the  desert  is  exposed  to  extreme  climatic  variations. 
It  is  not  any  single  factor  that  determines  the  features  commonly  ascribed 
to  the  desert,  but  rather  a  great  complex  of  conditions.  Thus,  while  scanty 
water-supply  may  be  of  greatest  importance,  it  is  not  only  the  low  average 
rainfall  which  makes  for  aridity,  but  the  distribution  of  this  precipitation 
throughout  the  year,  as  well  as  the  nature  of  the  soil  and  its  drainage.  To 
these  must  be  added  other  conditions,  such  as  the  low  relative  humidity  of 
the  air  and  the  high  winds,  all  favorable  conditions  for  water-loss,  great 
diurnal  variation  in  temperature,  and  intense  sunlight 

In  table  11  is  given  the 
monthly  precipitation  during 
1916  and  1917,  the  years  in 
which  these  investigations  were 
carried  out. 

In  this  consideration  rainfall 
is,  of  course,  of  importance  only 
as  it  replenishes  the  moisture  of 


TABLE  11. 


Month. 

1916. 

1917. 

Month. 

1916. 

1917. 

Jan.. 

3.94 

1.64 

July. 

3.37 

3.33 

Feb.. 

1.04 

0.43 

Aug. 

2.60 

1.84 

Mar. 

0.43 

0.14 

Sept. 

1.18 

1.19 

Apr. 
May. 

0.68 
0.00 

0.01 
0.73 

Oct.. 
Nov. 

1.48 
0.00 

0.00 
0.00 

June 

trace 

0.05 

Dec.. 

0.78 

0.00 

the  soil  as  a  supply  for  the  plant. 
Cannon *  has  shown  that  the  absorbing  roots  of  these  cacti  lie  at  a  depth  of 
10  to  20  cm.  It  is,  therefore,  rather  a  consideration  of  soil  moisture  at 
this  depth  which  is  of  immediate  importance.  Shreve  *  has  analyzed  the 
relation  between  rainfall  and  soil  moisture  in  the  region  from  which  the 
plants  used  in  the  present  investigation  were  taken.  There  are  two  well- 
defined  periods  of  rainfall  during  the  year — the  winter  rains,  December  to 
March,  and  the  summer  rains,  July  to  September.  For  the  34  years'  rain- 
fall record  of  Tucson,  Shreve  calculated  the  distribution  of  rainfall  during 
the  two  rainy  seasons  and  the  intervening  time.  The  percentages  of  the 
annual  total  are:  winter  31.7  per  cent;  arid  fore-summer  5.9  per  cent; 
humid  mid-summer  50.7  per  cent;  arid  autumn  11.7  per  cent.  The  sea- 
sonal variation  of  soil  moisture  during  the  year  runs  closely  parallel  to  the 
significant  periods  of  precipitation.  The  same  conclusion  may  be  arrived 
at  from  a  study  of  the  graphs  prepared  by  Livingston  *  of  the  relation  of  soil 
moisture  to  desert  vegetation.  This  well-defined  seasonal  distribution  of 
precipitation  gives  to  the  vegetation  a  very  marked  periodicity  of  activity.4 
Many  of  the  reactions  which  have  been  observed  as  characteristic  of  these 
seasons  have  their  origin  in  the  more  deep-seated  metabolic  activities  in- 

1  CANNON,  W.  A.    The  root  habits  of  desert  plants.    Carnegie  Inst  Wash.  Pub.  No. 

131,  43-52,  1911. 
1  SHBEVE,  F.    Rainfall  as  a  determinant  of  soil  moisture.    The  Plant  World,  17,  9-26, 

1914. 
1  LIVINGSTON,  B.  E.    Relation  of  soil  moisture  to  desert  vegetation.    Bot.  Gaz.,  50, 

241-256,  1910. 
4  MAcDouGAL,  D.  T.     The  course  of  the  vegetative  seasons  in  southern  Arizona.    The 

Plant  World,  11,  189-201,  217-231,  237-249,  261-270,  1908. 


SEASONAL  VARIATIONS  IN   THE  CARBOHYDRATE-CONTENT.     49 


duced  by  changes  in  the  external  conditions.  The  driest  portion  of  the 
year  is  the  arid  fore-summer  (April  to  July)  with  no  significant  rainfall, 
high  evaporation,  and  increasing  temperature.  The  arid  autumn  (October 
and  November)  is  as  dry  as  the  fore-summer,  but,  as  will  be  seen,  the 
effects  of  this  season  on  the  carbohydrate  economy  are  somewhat  moderated 
by  the  lower  temperatures  of  the  nights. 

In  table  12  are  given  the  maximum  and  minimum  temperatures  of  the 
locality  in  which  the  plants  investigated  grew. 

TABLE  12. 


Month 

19 

16. 

19 

17. 

19 

US. 

19 

L7. 

Max. 

Min. 

Max. 

Min  . 

Max. 

Min. 

Max. 

Min. 

Jan  
Feb 

•c. 

24.1 
27  2 

°c. 

-3.2 
-3  2 

•c. 

22.8 
26  6 

•c. 
0.2 
2  0 

Julv  
Aucr 

•c. 

43.8 
38  8 

•c. 
17.7 
15  4 

•c. 

32.2 
39  4 

•c. 

15.6 
15  4 

Mar  

33.2 

0.3 

28  1 

-0.9 

Sept. 

38  7 

16.4 

39.7 

16.4 

Apr  

32.5 

3.6 

34  2 

2.1 

Oct. 

35.0 

10.4 

38  5 

11.1 

May  
June  

38.3 
43.2 

10.0 
15.5 

36.4 
43.3 

6.6 
13.0 

Nov  
Dec  

34.2 
28.3 

0.2 
-2.1 

30.5 
27.1 

6.9 
2.0 

More  instructive  than  the  air  temperatures  are  the  temperatures  of  the 
cactus  joints  themselves.  Some  results  on  this  point  have  been  obtained  by 
McGee,1  and  while  these  are  not  complete  throughout  the  year,  an  idea  can 
be  obtained  therefrom  of  the  relation  of  air  temperatures  to  that  prevail- 
ing within  the  isolated  plant  McGee  summarizes  his  results  as  follows : 

"  (1)  Joints  of  Opuntia  blakeana  in  any  position  show  temperatures  above 
the  air-temperature  while  exposed  to  solar  radiation. 

"  (a)  The  temperatures  of  joints  in  an  equatorial  position  rise  steadily  till 
12  m.,  then  more  slowly  till  2  p.  m.,  when  the  maximum  is  reached.  After 
2  p.  m.  the  temperatures  steadily  decline,  becoming  the  same  as  that  of  the  air 
soon  after  sunset  and  then  falling  slightly  below  the  air-temperature  and 
remaining  so  during  the  night. 

"  (&)  The  temperatures  of  the  joints  in  a  meridional  position  rise  sharply 
after  sunrise,  reaching  a  maximum  about  11  a.  m.  They  then  slowly  drop  until 
12h  30m  p.  m.,  when  they  begin  to  rise  again,  reaching  the  second  and  highest 
maximum  point  about  4  p.  m.,  after  which  they  fall,  at  first  slowly  and  then 
more  abruptly,  till  sunset.  After  sunset  the  temperatures  slowly  fall  below  the 
air-temperature,  as  in  the  case  of  the  other  joints. 

"  (c)  Computation  of  the  area  inclosed  by  each  curve,  using  the  10-degree 
line  as  a  base,  shows  that  on  March  9,  1916,  the  number  of  hour-degree  units 
inclosed  by  the  air-temperature  curve  was  134.6 ;  by  the  curve  of  the  joints  in  an 
equatorial  position  211.5  hour-degree  units;  and  by  the  curve  of  the  joints  in  a 
meridional  position  230.8  hour-degree  units.  Hence  it  will  be  seen  that  the 
temperature  of  the  joints  in  a  north-and-south  position  exceeds  that  in  an 
east-and-west  position  by  19.3  hour-degree  units  and  the  air-temperature  by 
96.2  hour-degree  units,  and  that  in  these  joints  the  temperature  effects  would 
be  accentuated.  Similar  computations  show  that  on  June  2,  1916,  the  number 

1  MCGEE,  J.  M.    The  effect  of  position  upon  temperature  and  dry  weight  of  joints  of 

Opuntia.    Carnegie  Inst.  Wash.  Year  Book,  15,  73,  1916. 
4 


50  THE  CARBOHYDRATE  ECONOMY   OF  CACTI. 

of  hour-degree  units  inclosed  by  the  air-temperature  curve  was  273.0;  by  the 
curve  of  the  equatorial  joints  328.8  hour-degree  units ;  and  by  the  curve  of  the 
meridional  joints  376.9  hour-degree  units.  The  meridional  joints  exceed  the 
equatorial  joints  by  48.1  hour-degree  units  of  exposure,  and  the  air-temperature 
by  103.9  hour-degree  units. 

"  (d)  From  the  data  just  given,  it  will  be  seen  that  from  sunrise  to  sunset 
the  number  of  hour-degree  units  inclosed  by  the  temperature  curve  for  a  June 
day  is  very  much  greater  than  the  number  for  a  March  day,  and  that  the 
increase  is  greater  in  the  case  of  the  meridional  joints  than  in  that  of  the 
equatorial  joints.  The  numbers  of  hour-degree  units  inclosed  by  the  curves  of 
the  meridional  joints  for  March  9  and  for  June  2,  1916,  differ  by  146.1  hour- 
degree  units ;  the  numbers  inclosed  by  the  curves  of  the  equatorial  joints  differ 
by  117.3  hour-degree  units;  and  the  numbers  inclosed  by  the  curves  of  the  air- 
temperatures  differ  by  138.4  hour-degree  units. 

"  (e)  The  loss  of  weight  from  February  28,  1916,  to  April  5,  1916,  of  joints 
fn  a  meridional  position  was  18.59  per  cent,  the  loss  of  weight  of  joints  in  an 
equatorial  position  was  16.30  per  cent,  and  that  of  shaded  joints  was  5.79  per 
cent,  whereas  the  loss  of  weight  from  May  15  to  June  28,  1916,  of  joints  in  a 
meridional  position  was  24.70  per  cent,  that  of  joints  in  an  equatorial  position 
was  26.23  per  cent,  and  that  of  shaded  joints  was  23.32  per  cent.  The  dry 
weight  of  joints  similar  to  those  used  in  these  observations  was  16.15  per  cent 
on  March  8, 1916,  and  had  increased  to  17.70  per  cent  on  April  5,  an  increase  of 
1.55  per  cent;  whereas  the  dry  weight  of  joints  on  May  17  was  29.37  per  cent 
and  had  increased  36.38  per  cent  on  July  10,  1916,  an  increase  of  5.01  per  cent. 

"  (/)  The  maximum  temperatures  reached  by  joints  growing  under  natural 
conditions  were  found  to  be  53.0°  C.  on  July  24,  and  55.0°  C.  on  July  25,  1916. 
These  temperatures  are  higher  by  several  degrees  than  those  reported  by 
Askenasy  or  Ursprung  for  succulent  plants  such  as  Opuntia,  and  it  is  interesting 
to  note  that  Pfeifer  states  that  '  Prolonged  exposure  to  a  temperature  of  from 
45°  C.  to  46°  C.  kills  most  Phanerogams'  (Pfeffer's  Plant  Physiology,  vol  11. 
p.  226)." 

It  is  evident  from  these  results  that  the  conditions  of  temperature  are 
accentuated  within  the  plant  during  the  arid  fore-summer  with  its  clear 
days  of  intensive  insolation.  During  the  seasons  of  rain,  there  is,  of  course, 
relatively  much  less  direct  insolation  on  account  of  cloudy  and  overcast 
weather  and  this  would  result  in  correspondingly  lower  temperatures  of  the 
plant. 

To  summarize,  then,  the  period  December  to  March  is  characterized  by 
the  winter  rains  and  low  temperatures ;  the  period  April  through  June  by 
great  desiccation  and  high  temperatures ;  in  July  and  August,  the  humid 
mid-summer,  there  occur  heavy  rains  and  slightly  lower  temperatures, 
while  in  part  of  September,  October,  and  November,  the  arid  autumn,  there 
is  little  significant  rainfall  and  the  temperature,  while  still  high  in  the 
beginning  of  the  period,  moderates  in  October  and  November.  The  limits 
of  these  periods  may,  of  course,  extend  in  either  direction  and  vary  some- 
what in  different  years. 

In  this  section  are  given  the  results  of  a  study  of  the  seasonal  variation 
in  carbohydrate-content  of  Opuntia  phasacantha,  with  a  view  to  establishing 
the  nature  and  rate  of  its  metabolic  rearrangements  and  disintegration  as 
affected  by  the  climatic  conditions  just  discussed.  For  each  series  of 
analyses  the  joints  of  the  same  age  were  taken  from  a  single  large,  healthy 


SEASONAL  VARIATIONS  IN   THE  CARBOHYDRATE-CONTENT.     51 


plant.  It  was  found  that  individual  plants  vary  considerably  in  their  sugar- 
content,  although  the  general  course  of  the  economy  runs  parallel.  The 
methods  of  the  preparation  of  the  material  and  analysis  have  already  been 
described.  In  table  13  are  given  the  results  of  analyses  of  Opuntia  phcea- 
cantha  during  the  months  indicated  in  1916. 

TABLE  13. — Seasonal  variation  in  carbohydrate-content.    Analyses  expressed  in  per 
cent  of  the  dry  material. 


June  10. 

July  5. 

July  81. 

Sept.  20. 

Oct.  27. 

Nov.  15. 

Dec.  20. 

Water  

76.59 

63.62 

83  55 

80.34 

79  70 

76  95 

69  90 

Total  sugars  

32.62 

20  03 

13  24 

18.44 

20  90 

18  75 

18  95 

Total  polysaccharides.  . 

31  71 

19  35 

12.30 

17  80 

19  95 

16  82 

16  73 

Hexose  polysaccharides     .... 

16.05 

9.72 

8.16 

8.40 

8.86 

13.45 

6.04 

17  67 

10  45 

8  60 

8  83 

9  32 

5  50 

7  90 

Hexoses  plus  disaccharides..  . 
Disaccharides  

0.81 
0.36 

0.80 
0.18 

0.80 
0.14 

0.49 
0.20 

0.48 
0.10 

1.15 
0.39 

2.02 
1.04 

0  45 

0  62 

0  66 

0  29 

0  38 

0  76 

0  98 

Total  pentose  sugars  

14.95 

9.26 

4.39 

9.08 

10  95 

12.50 

10.45 

Pentosans  

14.81 

9.04 

4.14 

8.86 

10  47 

11.35 

10.10 

Pentoses  

0.14 

0.20 

0.25 

0.24 

0  48 

0.82 

0  35 

Monosaccharides  

0.59 

0.82 

0.91 

0  53 

0  86 

1.58 

1.33 

Although  the  values  given  in  table  13  indicate  the  nature  of  the  changes 
in  the  various  sugars  under  different  external  conditions,  the  facts  are  more 
clearly  expressed  as  proportional  values.  The  factor  of  the  synthesis  of 
the  carbohydrates  is  an  exceedingly  complex  one  and  can  not  be  dealt  with 
here,  so  that  variations  in  total  sugar-content  are  of  little  significance  for  the 
present  purpose.  The  influence  of  the  external  conditions  on  the  propor- 
tional values  will  be  brought  out  more  clearly  in  the  section  dealing  with 
the  special  influence  of  water-content  and  temperature  where  the  factor  of 
synthesis  plays  no  role,  as  the  plants  were  left  in  the  dark  and  at  constant 
temperature. 

In  table  14  the  results  of  table  13  are  given,  calculated  as  proportional 
values. 

TABLE  14. — Proportional  values  of  the  carbohydrate-content  of  Opuntia  phcvacantha 
on  the  basis  of  the  analysis  of  the  dry  material. 


June  10. 

Julys. 

July  31. 

Sept  .20. 

Oct.  27. 

NOT.  15. 

Dec.  20. 

Total  polysaccharides 

0.973 

0.960 

0.930 

0.965 

0.955 

0.897 

0.888 

Total  sugars 
Monosaccharides 

.019 

.042 

.074 

.039 

043 

.084 

.070 

Total  polysaccharides 
Hexoses  plus  disaccharides 

.051 

.082 

.098 

.058 

.054 

.085 

.335 

Hexose  polysaccharides 
Hexoses 

.025 

.064 

.081 

.035 

.043 

.056 

.162 

Hexose  polysaccharides 
Total  pentose 

.459 

.462 

.332 

.492 

.524 

.667 

.551 

Total  sugars 

52 


THE   CARBOHYDRATE  ECONOMY  OF   CACTI. 


This  series  runs  from  the  arid  fore-summer  through  the  humid  mid- 
summer, the  arid  autumn,  and  into  the  beginning  of  winter  with  its  higher 
rainfall  and  lower  temperatures.  What,  then,  are  the  changes  which  occur 
in  the  carbohydrate-content  of  these  cacti  when  exposed  to  these  decided 
changes  in  the  environment  ?  The  values  in  tables  13  and  14  for  June  repre- 
sent the  effect  of  extreme  desiccation  and  high  temperatures ;  there  fell  only 
0.68  inch  of  rain  in  the  preceding  April,  none  in  May,  and  but  a  trace  in 
June.  It  is  evident  that  here  the  polysaccharides,  both  in  actual  amount 
and  in  the  proportion  to  the  other  sugar  groups,  are  high.  The  mono- 
saccharides,  on  the  other  hand,  by  the  same  criteria,  are  low,  while  the 
pentosans  and  total  pentoses  are  relatively  high.  In  the  analysis  of  July  5 
the  desiccation  has  proceeded,  though  this  was  taken  immediately  after  the 
first  rain  and  the  effect  on  the  carbohydrates  is  hardly  perceptible.  By 
July  31,  the  next  analysis,  3.37  inches  of  rain  have  fallen:  the  water-con- 

TABLE  15. — Seasonal  variation  in  carbohydrate-content.    Analyses  expressed  in 
percentage  of  the  dry  material. 


J»n.  11. 

Feb.  16. 

Mar.17. 

Apr.  25. 

May  22. 

Oct.  31. 

Dec.  19. 

Jan.  24. 

Water 

77.80 
19.10 
15.10 
10.45 
14.95 
3.86 
1.27 
2.59 
4.73 
4.40 
0.43 
3.02 

77.90 
21.32 
15.80 
9.98 
14.90 
5.30 
1.74 
3.56 
6.07 
5.51 
0.55 
4.11 

80.50 
28.05 
20.10 
15.00 
22.16 
7.80 
0.93 
6.87 
5.55 
4.75 
0.82 
7.69 

75.70 
32.40 
29.84 
20.65 
22.70 
2.27 
0.41 
1.86 
9.15 
8.68 
0.48 
2.34 

74.75 
30.15 
28.38 
15.47 
17.08 
0.76 
0.48 
0.28 
12.34 
12.17 
0.16 
0.44 

72.75 
26.40 
24.95 
15.30 
16.61 
1.31 
0.48 
0.83 
9.79 
9.65 
0.14 
0.97 

66.75 
26.88 
21.14 
20.76 
25.12 
4.36 
1.88 
2.48 
1.76 
1.38 
0.38 
2.86 

66.10 
25.90 
21.35 
16.92 
21.40 
4.48 
2.18 
2.30 
4.50 
4.23 
0.27 
2.45 

Total  polysaccharides  

Hexose  polysaccharides  
Total  hexose  sugars  

Hexoses  plus  disaccharides.  .  . 
Disaccharides  

Hexoses                  

Total  pentose  sugars. 

tent  is  decidedly  higher,  though  the  temperatures  continue  high.  The 
polysaccharides,  both  in  actual  amount  and  in  proportion  to  the  other  sugar 
groups,  have  fallen.  The  monosaccharides  and  the  hexoses  are  higher  than 
during  the  dry  periods.  The  pentosans  show  a  marked  decrease  with  the 
increased  water-content.  The  September  analysis  represents  the  beginning 
of  the  arid  autumn,  there  has  been  little  rain,  and  the  water-content  has 
dropped.  The  total  polysaccharides  have  again  risen,  also  in  proportion  to 
the  other  sugars ;  the  monosaccharides  show  a  corresponding  drop,  and  the 
pentosans  have  also  risen.  During  the  following  months,  October,  Novem- 
ber, and  December,  the  drought  continues,  with  corresponding  low  water- 
content.  The  amount  of  the  polysaccharides  again  rises  during  October. 
In  November  and  December,  however,  in  spite  of  the  continued  dryness, 
the  relative  values  again  decrease.  So  also  the  monosaccharides,  while  very 
low  in  October,  increase  in  November  and  December.  The  pentosans  show 
a  constant  increase  until  they  drop  slightly  in  December.  It  appears,  there- 
fore, that  the  effects  of  the  drought  are  first  moderated  and  then  counter- 
acted by  the  lower  temperatures  prevailing  during  November  and  December. 


SEASONAL  VARIATIONS  IN   THE  CARBOHYDRATE-CONTENT.     53 


The  proportional  values  calculated  from  the  results  in  table  15  are  given 
in  table  16. 

Further  light  on  these  phenomena  can  be  gained  from  a  study  of  tables 
15  and  16,  giving  the  results  of  analyses  of  joints  from  the  same  plant  dur- 
ing 1917.  These  cover  the  periods  beginning  with  the  humid  winter,  then 
the  arid  fore-summer,  the  arid  autumn,  and  finally  again  the  humid  winter. 
The  humid  summer  is  not  included,  as  this  period  is  covered  in  the  pre- 
ceding series.  The  water-content  increases  during  January,  February,  and 
March,  though  the  rainfall  was  relatively  light  for  this  season.  Although 
the  total  amount  of  sugar  present  increases,  the  relative  amounts  of  the 
polysaccharides  decrease  with  increasing  water-content.  The  simpler 
sugars,  hexoses  or  monosaccharides,  show  again  a  corresponding  increase 
with  increased  water-content,  both  in  actual  amount  and  in  proportion  to 

TABLE  16. — Proportional  values  of  the  carbohydrate-content  of  Opuntia  phaacantha 
on  the  basis  of  the  analysis  of  dry  material. 


Jan.  11. 

Feb.  16. 

Mar.  17. 

Apr.  25. 

May  22. 

Oct.  31. 

Dee.  10. 

Jan.  24. 

Total  polysaccharides 

0.791 
.200 
.202 
.248 
.248 

0.741 
.260 
.249 
.357 
.283 

0.716 
.380 
.278 
.457 
.198 

0.907 
.078 
.070 
.090 
.283 

0.942 
.016 
.025 
.018 
.409 

0.945 
.039 
.049 
.054 
.371 

0.787 
.135 
.162 
.195 
.066 

0.821 
.115 
.173 
.136 
.174 

Total  sugars 
Monosaccharides 

Total  polysaccharidea 
Hexoses  plus  disaccharides 

Hexose  polysaccharides 
Hexoses 

Hexose  polysaccharides 
Total  pentose 

Total  sugars 

the  other  sugar  groups.  The  monosaccharides  thus  reach  their  maximum 
in  March  at  a  time  when  the  growth  activity  of  the  plant  commences.  The 
pentose  sugars  show  a  relative  decrease  with  the  higher  water-content. 
The  period  from  March  17  to  April  25  was  an  exceedingly  dry  one,  the 
water-content  has  dropped,  and  although  the  total  sugar-content  has  in- 
creased, the  proportion  of  these  sugars  is  very  different  from  what  it  was  at 
the  end  of  the  winter  rainy  season.  The  polysaccharides  have  again 
increased  in  every  respect,  the  monosaccharides  have  decreased  very 
decidedly,  and  the  pentosans  have  increased  markedly.  During  May  the 
arid  fore-summer  conditions  persist,  with  increased  temperatures,  and  the 
condition  of  the  carbohydrates  as  described  for  April  are  accentuated ;  very 
marked  is  the  increase  in  pentosans.  The  conditions  from  June  to  Sep- 
tember are  a  repetition  of  what  has  already  been  described  for  these  seasons. 
The  arid  autumn  brings  again  the  high  polysaccharide  and  low  mono- 
saccharide  content  with  abundance  of  pentosans.  With  the  falling  tem- 
peratures and  rain  of  winter,  the  reverse  conditions  again  set  in.  (The 
total  pentose  sugar  and  pentosan  value  for  December  is  not  reliable,  as  it 


54  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

was  discovered  that  an  impure  culture  of  yeast  had  been  used  for  the 
fermentation. ) 

To  summarize,  then,  the  observations  of  the  seasonal  variations  in  carbo- 
hydrate-content : 

Low  water-content  and  high  temperatures  are  associated  with:  (1)  in- 
crease of  polysaccharides ;  (2)  decrease  of  monosaccharides ;  (31)  increase 
of  pentosans. 

High  water-content  and  lower  temperatures  are  associated  with:  (1) 
decrease  of  polysaccharides ;  (2)  increase  of  monosaccharides ;  (3)  decrease 
of  pentosans. 

It  remains,  then,  to  determine  experimentally  whether  under  controlled 
conditions  such  a  shifting  of  the  relative  amounts  of  the  various  sugars 
actually  takes  place,  and  to  what  extent  the  two  factors,  water-content  and 
temperature,  contribute  to  such  an  action.  Before  turning  to  these  experi- 
ments, it  may  be  of  some  value  to  consider  briefly  some  other  observations 
pertinent  to  the  general  problem. 

During  March  the  new  joints  develop ;  these  grow  quite  rapidly,  so  that 
within  about  one  month  they  have  attained  their  full  size  of  100  to  125  cm. 
Apparently  the  young  joints  become  autonomous  very  early  in  their  develop- 
ment ;  when  cut  from  the  plant  with  but  a  very  small  portion  of  the  parent 
joint,  and  placed  with  the  base  in  tap-water,  the  young  joints  grow  to  full 
size  and  develop  normally.  Comparative  analyses  of  young  and  parent 
joints  are  given  in  table  17.  The  first  young  joints  (March  27,  1916)  were 
2  to  4  cm.,  about  12  days  old. 

The  young  joints  are  very  high  in  total  sugars  compared  with  the  parent 
joints.  Although  the  young  joints  have  attained  almost  their  full  size  by 
April  16,  the  percentage  of  cellulose  has  not  increased  and  the  joints  are 
very  tender,  while  in  the  old  joints  the  cellulose  is  considerably  higher,  due 
to  the  development  of  walls  and  vessels.  The  inorganic  constituents  increase 
rapidly  in  the  young  joints.  It  is  a  striking  fact  that  although  the  young 
joints  have  a  higher  total  sugar-content  than  the  parent  joints,  both  the 
actual  percentages  and  the  proportional  values  of  the  monosaccharides  are 
lower  in  the  former.  This  is  probably  due  to  the  high  respiratory  activity 
of  the  young  joints.  It  is  evident  that  the  amount  of  pentose  sugar  is  con- 
siderable, even  in  the  very  early  stages  of  development,  although  the  propor- 
tion to  the  total  sugars  is  somewhat  higher  in  the  old  joints. 

It  has  commonly  been  affirmed  that  the  pentosans  accumulate  in  the  older 
portions  of  a  plant.  This  contention  is  based  upon  results  obtained  from 
the  older  methods  of  analyses,  in  •  vhich  the  plant  material  was  treated  with 
hydrochloric  acid  of  such  concentrations  as  to  affect  also  the  cellulose.  As 
by  this  treatment  cellulose  also  yields  furfural,  the  formation  of  which  is 
taken  as  a  measure  of  the  pentosan  content,  it  is  not  surprising  that  the 
older  portions  of  a  plant,  which  are  very  generally  richer  in  cellulose,  should 
give  results  indicating  large  quantities  of  pentosans.  That  the  older  parts 
of  plants  are  not  always  richer  in  pentosans  than  the  portions  more  recently 
formed  is  shown  by  results  of  analyses  of  the  following  two  sets  of  joints. 


SEASONAL  VARIATIONS  IN   THE  CARBOHYDRATE-CONTENT.     55 


TABLE  17. — Carbohydrate-content  of  young  and  parent  joints  of  Opuntia  phwacantha. 
Values  given  in  percentages  of  the  dry  material. 


Mar.  27,  1916. 

Apr.  3. 

Apr.  16. 

Young. 

Parent. 

Young. 

Parent. 

Younr. 

Parent. 

Water  

88.65 
31.22 
2.74 
6.23 
7.24 
6.88 

0.088 
0.199 

81.38 
22.44 
3.22 
5.21 
11.35 
12.60 

0.144 
0.232 

87.30 
33.60 
3.37 
9.57 
6.96 
7.51 

0.100 
0.285 

81.10 
20.18 
5.32 
10.04 
11.86 
14.40 

0.264 
0.495 

88.40 
36.68 
4.43 
7.48 
7.22 
10.71 

0.121 
0.204 

81.78 
28.40 
5.00 
5.23 
12.08 
13.95 

0.176 
0.185 

Total  sugars            

Monosaccharides  

Total   pent  ose  

Cellulose  

Ash  

Monosaccharides 

Total  sugars 
Total  pentoses 

Total  sugars 

The  analyses  were  made  in  October.  From  the  same  plant  were  taken  a 
number  of  joints  formed  in  the  spring  of  the  year  1916,  as  well  as  a  number 
of  joints  which  had  been  formed  three  years  or  more  previous,  as  was 
indicated  by  their  position  on  the  plant;  i.  e.,  the  old  joints  were  fourth  in  a 
row  counting  from  the  youngest  joints.  The  results  are  given  in  table  18. 

TABLE  18. — Carbohydrate-content  of  young  and  old  joints  of  Opuntia  phceacantha, 
expressed  in  percentages  of  dry  material. 


Joint  formed  in 

Joint  formed  in 

1916. 

1913. 

1916. 

1913. 

Water 

82.15 
20.45 
17.94 
10.10 
1.18 

79.89 
16.60 
14.45 
9.10 
0.51 

1.27 

9.78 
0.23 
9.55 
9.10 

1.22 
7.10 
0.40 
6.70 
10.26 

Total  sugars  

Total  pentose  

Total  polysaccharides  
Total  hexose  sugars  

Pentoses  

Pentosans  

Disaccharides.         

Cellulose  

Although  the  cellulose  is  higher,  it  is  noticeable  that  the  pentosan  content 
is  lower  in  the  old  joints.  This  was  found  in  a  number  of  other  analyses 
as  well.  In  general,  the  carbohydrate  content  of  the  younger  joints  is 
higher  than  in  older  ones,  though  the  proportion  of  the  various  groups  of 
sugars  to  the  total  sugars  is  about  the  same  in  the  two  sets  of  joints. 

As  yet  it  has  not  been  possible  to  obtain  entirely  satisfactory  results  on 
the  photosynthetic  activity  of  the  mature  cacti.  The  complexity  of  the  gas 
interchange  and  the  influence  thereon  of  the  high  acidity  as  described  by 
Richards,  has  already  been  alluded  to.  The  diurnal  variation  in  carbo- 
hydrate content  is  slight  in  these  plants.  In  table  19  are  given  the  results 
of  analyses  made  before  and  after  insolation. 

The  loss  of  water  during  the  night,  resulting  in  an  increase  of  the  total 
dry  weight,  is  evident.  This  is  a  phenomenon  commonly  observed  in  these 
plants.  The  increase  of  the  disaccharides  and  monosaccharides,  both  in 
actual  amount  and  in  the  proportion  to  the  polysaccharides,  is  considerable. 


56 


THE   CARBOHYDRATE  ECONOMY   OF   CACTI. 


How  much  of  this  increase  is  due  directly  to  the  photosynthetic  activity  it  is 
difficult  to  establish,  as  the  independent  influence  of  the  changed  water- 
content  on  the  proportion  of  simple  sugars  to  polysaccharides  in  all  prob- 
ability comes  into  play.  This  will  be  discussed  in  the  following  section. 

TABLE  19. — Variation  in  carbohydrate-content  of  Opuntia  phceacantha  during 
24  hours.    Results  in  percentages  of  the  dry  material. 


5p.m. 

7»30»»  a.  m. 

5p.m. 

Dry  weight  ....         

17.58 

18.80 

17.85 

Total  sugars  

16.18 

19.40 

20.45 

Total  polysaccharides  

14.40 

18.15 

17.94 

0.58 

0.34 

1.18 

Hexoses 

1.06 

0.83 

1.27 

8.60 

8  33 

9.78 

8  34 

8  22 

8.55 

Pentoses  

0.26 

0.20 

0.23 

Monosaccharides 

0  092 

0  057 

0  C84 

Total  polysaccharides 

In  perusing  the  results  of  the  seasonal  variation  in  carbohydrate-content, 
of  significance  is  the  fact  that  the  greatest  activity  of  the  plant  comes  at 
the  time  when  the  content  of  monosaccharides  and  disaccharides  is  highest 
The  vegetative  shoots  and  flowers  are  developed  in  March,  and  it  is  at  this 
time  that  the  general  process  of  inversion  has  reached  its  maximum.  This 
process  of  carbohydrate  inversion,  taking  place  in  the  plant  as  a  consequence 
of  low  temperature  and  ample  water-supply,  can  be  prevented  by  artificially 
keeping  the  plant  either  at  a  higher  temperature  or  with  a  low  water-supply. 
In  neither  case  does  the  plant  then  develop  vegetative  or  flower  buds  at  the 
regular  season.  Thus,  a  number  of  plants  of  Opuntia  ph&acantha  were  put 
in  soil,  and  so  placed  out  of  doors  that  they  obtained  only  a  minimum 
amount  of  rainfall  during  the  winter  rainy  season.  These  plants  produced 
no  flowers  or  vegetative  shoots.  Other  plants  similarly  treated,  but  allowed 
to  receive  the  normal  rainfall,  developed  normally  in  the  spring.  Again, 
a  number  of  plants  were  kept  at  higher  temperatures  than  normal  by  cover- 
ing them  with  glass  cages.  Through  the  "  trapping  "  of  the  solar  radiation 
thus  effected,  the  temperatures  within  the  cages  were  decidedly  higher  than 
the  surrounding  air.  The  cages  were  not  large  enough  to  prevent  the  rain 
from  reaching  the  extensive  root  system.  None  of  the  plants  thus  treated 
produced  either  flowers  or  new  joints  in  the  spring.  Similarly,  plants  which 
had  been  kept  in  glass  cages  artificially  heated  during  the  winter  showed  the 
same  behavior.  It  can  not  be  maintained,  of  course,  that  the  accumulation 
of  relatively  large  quantities  of  the  simpler  sugars  is  the  only  prerequisite 
to  growth.  It  is  not  possible  to  indicate  any  single  factor  or  substance  to 
which  can  be  ascribed  such  properties,  nor  is  such  a  state  of  affairs  likely  to 
exist  Physiological  activity  in  all  probability  represents  the  "  resultant 
of  forces  "  and  a  supply  of  simple  sugars  above  that  required  for  the  normal 
respiratory  activity  seems  to  be  one  of  the  factors  necessary  for  growth. 


EFFECT   OF  WATER  ON  THE  CARBOHYDRATE-CONTENT.        57 


VI.    EFFECT  OF  WATER  ON  THE  CARBOHYDRATE- 
CONTENT. 

It  has  already  been  pointed  out  that  the  platyopuntias  respond  quickly 
to  available  water,  and  that  although  the  rate  of  water-loss  is  low,  the  plant 
is  capable  of  undergoing  relatively  great  fluctuations  in  its  water-content. 
Under  natural  conditions  these  plants  are  exposed  alternately  to  conditions 
of  extreme  drought  and  to  abundant  water-supply.  The  changes  in  the 
carbohydrate-content  occur  especially  at  the  times  of  the  well-defined  periods 
of  rainfall.  Low  temperatures  influence  the  carbohydrates  in  the  same 
manner  as  high  water-content  does;  both  result  in  a  condition  of  general 
inversion.  It  was  necessary,  therefore,  to  determine  independently  the 
influence  of  changes  in  water-content  on  the  carbohydrate  equilibrium.  For 

TABLE  20. — Loss  of  weight  of  joints  of  Opuntia  phceacantha 
in  a  humid  atmosphere. 


Date. 

Days. 

Weight. 

Loss  in 
grama. 

Percentage 
of  loss. 

Feb.  21  

397 

Mar.    1  

8 

396 

1 

0.4 

Mar.  12  

19 

393 

4 

1.0 

Apr.    3  

41 

391 

6 

1  5 

Apr.    6  

44 

391 

6 

1.5 

TABLE  21. — Loss  of  weight  of  joints  of  Opuntia  phceacantha 
in  a  dry  atmosphere. 


Date. 

Days. 

Weight. 

Loss  in 

grams. 

Percentage 
of  loss. 

Feb.  21  

365 

Mar.    1   

8 

340 

25 

6.8 

Mar.   5  

12 

330 

35 

9.6 

Mar.  12  

19 

320 

45 

12.3 

Apr.    3...     . 

41 

297 

68 

18.6 

Apr.    6  

44 

294 

71 

19.5 

this  purpose  a  number  of  healthy  joints  of  the  same  age  were  cut  from  the 
same  plant.  These  were  divided  into  three  sets  of  seven  joints  each ;  one 
set  was  analyzed  immediately,  another  set  was  placed  under  a  large  bell-jar 
over  a  dish  of  water,  and  the  third  set  was  placed  under  a  similar  bell-jar 
over  a  dish  containing  anhydrous  calcium  chloride.  A  slow  stream  of  moist 
and  dry  air  respectively  was  passed  through  the  bell-jars.  These  were  kept 
in  the  dark  in  a  constant-temperature  chamber  at  20°  C.  The  experiment 
ran  for  44  days ;  at  intervals  the  plants  were  weighed  to  follow  the  loss  of 
weight.  In  table  20  the  results  of  these  weighings  are  given. 

Thus  the  joints  which  were  left  in  a  humid  atmosphere  lost  in  44  days 
only  6  grams  or  1.5  per  cent  of  their  total  weight  At  the  end  of  this  time 
the  water-content  had  fallen  from  81.5  to  81.2  per  cent.  The  joints  kept 
over  calcium  chloride  lost  much  more  water,  as  is  shown  in  table  21. 


58 


THE   CARBOHYDRATE  ECONOMY   OF   CACTI. 


After  44  days  of  desiccation  these  plants  lost  71  grams  or  19.5  per  cent  of 
their  original  weight  At  the  end  of  the  experiment  the  water-content  was 
YT.O  per  cent,  which,  of  course,  does  not  equal  the  extreme  desiccation  the 

TABU:  22. — Carbohydrate-content  of  Opuntia  phceacantha  before  and  after  44  days  in 
the  dark  at  20°  C.  in  percentages  of  the  dry  material. 


Origi- 

Origi- 

nal 
condi- 

Kept 
dry. 

Kept 
moist. 

nal 
condi- 

Kept 
dry. 

Kept 
moist. 

tion. 

tion. 

Water 

81  50 

77  00 

81  20 

3  47 

0  60 

0  58 

26  83 

22  08 

23  76 

3  47 

1  84 

2  30 

Total  polysaechar  idea  . 

19.02 

19.40 

20.47 

Monosaccharides  

4.34 

2.08 

2.71 

Disaccharides    plus 

Total  pentoses  

1.70 

1.85 

1.37 

hexoees    

7.21 

2.44 

2.88 

Pentoses  

1.60 

0.24 

0.41 

Total  hex  use  sugars 

25  13 

20.23 

22.39 

Pentosans  

1.10 

1.61 

0  96 

Hexose  polysaccharides 

17.92 

17.79 

19.52 

plants  attain  under  natural  conditions.  The  processes  which  take  place 
during  such  a  period  of  desiccation  are,  of  course,  by  no  means  simple,  and 
it  can  not  be  considered  as  a  simple  loss  of  water.  In  the  first  place,  the 
factor  of  respiration  or  the  consumption  of  carbohydrates  is  to  be  taken  into 
consideration.  If  it  is  assumed  that  the  reaction  is  of  the  empirical  nature 
C6H12O6  +  6O2-^6H*O  -f  6CO2,  there  is  formed  for  each  gram  of  sugar 
burned  0.6  gram  of  water,  which  can  be  considered  as  remaining  within  the 
plant.  For  each  gram  of  total  loss  in  weight  of  the  plant  due  to  carbohy- 
drate burning,  2.5  grams  of  sugar  must  be  consumed,  resulting  in  the 
formation  of  1.5  grams  of  water.  Thus  the  respiration  of  the  plant  tends 
not  only  to  reduce  the  dry  mass  but  as  well  to  increase  the  water-content. 
A  second  complicating  factor  is  introduced  when  it  is  considered  that  the 

TABLE  23. — Proportional  values  of  carbohydrate-content  of  Opuntia  phceacantha 
before  and  after  44  days  in  the  dark  at  20°  C. 


Original 
condition. 

Kept  dry. 

Kept  moist. 

Total  polysaccharides 

0.709 

0.879 

0.860 

Total  sugars 
Hexose 

0.209 

0.104 

0.118 

Hexose  polysaccharides 
Monosaccharides 

0.228 

0.107 

0.133 

Total  polysaccharides 
Hexoses  plus  disaccharides 

0.405 

0.137 

0.148 

Hexose  polysaccharides 
Pentosans 

0.041 

0.073 

0.041 

Total  sugars 

hydrolysis  of  polysaccharides  takes  place  accompanied  by  the  absorption  of 
water.  This  water  is  incorporated  in  the  chemical  composition  of  the 
sugars.  The  withdrawal  of  water  by  this  process  tends  to  increase  the  dry 


EFFECT   OF  WATER  ON   THE   CARBOHYDRATE-CONTENT.        59 


mass  as  well  as  to  decrease  the  water-content.  These  factors  are  unques- 
tionably of  great  importance  to  the  plant,  especially  under  conditions  of 
restricted  water-supply  and  during  starvation.  Discussion  hereof  will  be 
taken  up  again  in  a  following  section. 

Table  22  gives  the  results  of  analyses  of  the  three  sets  of  cactus  joints. 

The  proportional  values  calculated  from  table  22  are  given  in  table  23. 

The  water-content  of  the  plants  kept  over  calcium  chloride  was  reduced 
from  81.5  per  cent  to  77.0  per  cent;  those  kept  over  water  were  reduced  to  a 
water-content  of  81.2  per  cent.  In  both  cases  there  was  a  decided  reduction 
in  total  sugars.  From  the  available  data  it  is  impossible  to  tell  whether  all 
of  this  reduction  is  due  to  respiration  or  whether  in  part  it  is  due  to  the 
formation  of  carbohydrates  of  the  nature  of  cellulose  which  are  not  hydro- 
lyzed  with  1  per  cent  by  hydrochloric  acid  and  hence  do  not  appear  in  the 
mass  of  total  sugars.  It  is  a  noteworthy  fact,  however,  that  the  actual 
percentage  of  total  polysaccharides  shows  an  increase  in  both  cases.  There 
is  very  little  difference  in  the  rates  of  respiration  between  the  two  sets  of 
plants,  as  is  shown  in  table  24. 

TABLE  24. — Rate  of  respiration  in  milligrams  C02  of  joints  of  Opuntia  phceacantha 
after  "being  kept  in  the  dark  for  44  days. 


Original 
condition. 

Kept  dry. 

Kept  moist. 

CO2  per  gram  fresh  weight  per  hour.             

0  0047 

0  0038 

0  0033 

0  0252 

0  0165 

0  0176 

0  6730 

0  8970 

0  7650 

From  results  given  in  tables  22  and  23  it  is  clear  that  under  controlled 
conditions  the  comparative  values  of  the  carbohydrate-content  show  that 
with  decreased  water-content  the  monosaccharides  exhibit  a  decrease  while 
the  polysaccharides  show  an  increase.  Also  the  pentosans  increase  with 
decreasing  water-supply. 

TABLE  25. — Carbohydrate-content  of  Opuntia  phceacantha  as  affected  by  increase  and 
decrease  in  water-content.    Values  given  in  percentages  of  the  dry  material. 


Origi- 
nal con- 
dition 

Kept 
dry. 

SI 

Origi- 
nal con- 
dition. 

Kept 
dry. 

moist. 

Water 

80  34 

77  20 

82  30 

Total  sugars  

18.44 

16.72 

16.94 

char  ides  

0.49 

0.53 

0.79 

Total  polysaccha- 

Disaccharides  

0.20 

0.29 

0.36 

17.80 

15.96 

16.04 

Hexoses   

0.29 

0.26 

0.45 

Total  pentoses  

9.08 

8.42 

8.02 

8  40 

7.31 

7.70 

Pentoses  

0.24 

0.23 

0.17 

8  83 

7.81 

8.50 

Pentosans.         

8.86 

8.19 

7.85 

Similar  results  are  obtained  when  instead  of  allowing  only  one  set  of  the 
cactus  joints  to  lose  water,  the  experiment  is  so  arranged  that  one  set  loses 
water  while  another  exactly  similar  increases  in  water-content  This  was 
done  by  placing  one  set  of  seven  joints  in  glass  battery  jars  in  such  a  manner 


60  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

that  the  cut  end  of  the  joint  extended  into  tap-water.  It  was  found  neces- 
sary to  allow  these  cut  surfaces  to  dry  out  well  before  placing  in  water ;  in 
this  way  any  diffusion  of  sugar  from  the  plant  into  the  water  was  avoided. 
Another  similar  set  of  joints  was  placed  in  the  same  kind  of  vessel  without 
water.  Within  a  week  the  joints  in  water  formed  thin  roots,  which  grew 
15  to  20  cm.  in  length  within  the  month,  but  no  trace  of  sugar  was  found  in 
the  water  at  the  end  of  this  time.  The  experiment  was  carried  out  in  the 
dark  at  constant  temperature  of  20°  C.  The  analyses  are  given  in  table  25. 

From  the  experimental  results  just  presented  it  appears  that  the  con- 
clusions in  regard  to  the  effect  of  water  on  the  carbohydrate-content  given 
in  the  preceding  section  are  justified. 

The  agents  effecting  these  changes  of  inversion  and  reversion  of  the 
carbohydrates  according  to  the  varying  conditions  of  water-supply  are  not 
definitely  known.  The  present  state  of  our  knowledge  of  reversible  enzyme 
action  offers  little  enlightenment  on  the  phenomena  under  discussion.  An 
explanation  may  be  ventured,  however,  based  on  the  known  properties  of  the 
plasmic  colloids.  One  of  the  most  striking  properties  of  hydrophile  colloids 
is  the  avidity  with  which  they  take  up  water  and  the  great  pressure  de- 
veloped in  the  process.  Conversely,  also,  it  requires  enormous  pressures  to 
express  imbibed  water,  the  colloids  holding  the  water  with  tremendous 
tenacity.1  Contrary  to  the  water  in  solutions  of  crystalloidal  salts,  the  water 
in  these  colloidal  dispersions  is  firmly  held  and  has  been  designated  as 
"  hydratation  water."  Evidence  of  the  avidity  with  which  these  colloids  take 
up  water  can  be  gained  from  the  fact  that  they  absorb  water  and  swell  from 
concentrated  solutions  of  salts  such  as  calcium  nitrate  and  ammonium 
sulphate.  Now,  the  inversion  of  polysaccharides  involves  the  taking  up  of 
water  into  the  chemical  composition  of  the  resulting  simpler  carbohydrate. 
For  each  molecule  of  polysaccharide  inverted  or  hydrolyzed,  at  least  one 
molecule  of  water  is  taken  up.  As  the  water-content  of  the  plant  diminishes, 
the  available  water  for  this  process  is  reduced.  If  finally  the  water  is  so 
firmly  held  that  it  can  not  enter  into  the  chemical  process  of  inversion,  the 
hydrolysis  of  the  polysaccharides  can  not  take  place,  or,  what  is  more  prob- 
able, the  inversion  takes  place  at  a  greatly  reduced  rate.  It  must  soon  be 
realized  that  existing  chemical  knowledge  is  not  sufficient  to  interpret  the 
reactions  taking  place  in  living  organism.  As  was  emphasized  in  the  intro- 
ductory discussion,  these  reactions  do  not  take  place  under  conditions  of 
pure  aqueous  solution,  and  deductions  based  upon  such  assumptions  must 
prove  inadequate  in  considering  the  chemical  changes  in  living  things. 
Some  knowledge  of  reactions  in  colloidal  media  must  supplement  the 
existing  physical-chemical  conceptions.  The  formation  of  pentosans  under 
conditions  of  decreased  water-content,  as  pointed  out  previously,  is  inti- 
mately associated  with  the  reduced  supply  of  monosaccharides.  These 
phenomena  will  be  discussed  under  the  section  on  pentose  sugars. 

1  REINKE,  J.    Untersuchungen  ueber  die  Quellung  einiger  vegetablischer  Substanzen. 
Haustein's  Bot.  Abt.,  4,  Heft  1. 


EFFECT  OF  TEMPERATURE  ON  THE  CARBOHYDRATE-CONTENT.  61 

VII.  EFFECT  OF  TEMPERATURE  ON  THE 
CARBOHYDRATE-CONTENT. 

The  influence  of  temperature  on  the  carbohydrate-content  of  plants  such 
as  the  platyopuntias  is,  of  course,  the  summation  of  the  effect  of  this  factor 
on  a  variety  of  activities.  In  order  to  eliminate  photosynthetic  activity  in 
the  tests,  the  plants  were  kept  in  the  dark.  In  general  the  experiments  were 
carried  out  in  the  same  manner  as  those  described  in  the  preceding  section. 
For  each  test  a  number  of  joints  of  the  same  age  were  taken  from  a  single 
healthy  plant;  one  set,  usually  seven  joints,  was  immediately  analyzed, 
while  other  sets  were  subjected  to  experimental  conditions.  Whether  the 
increase  of  disaccharides  and  monosaccharides  is  the  result  of  the  effect  of 
lower  temperatures  on  certain  enzyme  actions,  or  whether  this  merely  repre- 
sents an  accumulation  of  these  sugars  due  to  the  reduced  respiratory  activity, 
it  is  difficult  to  establish  quantitatively.  In  considering  the  effect  of  tem- 
perature on  the  seasonal  variations  of  the  carbohydrates  in  the  cacti,  it  must 
be  borne  in  mind  that  during  the  warm  seasons  the  high  temperatures  are 
maintained  for  a  long  duration.  It  is  this  factor  which  must  be  of  special 
importance  in  applying  the  thermolabile  properties  of  the  enzymes  to  the 
present  problem.  Relatively  high  temperatures  may  be  borne  by  many 
enzymes  for  short  periods,  but  when  such  temperatures  are  maintained  for 
longer  periods  rapid  inactivation  ensues.  Davis1  has  shown  that  maltase  in 
plants  is  greatly  reduced  in  activity  and  finally  destroyed  at  temperatures 
above  50°.  From  the  records  of  McGee  on  the  temperatures  in  cacti, 
already  referred  to,  it  is  evident  that  these  plants  during  the  summer  attain 
35°  and  higher,  and  that  these  temperatures  are  maintained  during  the  day 
for  8  to  10  hours.  A  temperature  of  50°  within  the  plant  is  not  at  all 
uncommon.  Such  conditions  may  persist  daily  for  5  months. 

An  indication  of  the  effect  of  temperature  on  the  rate  of  respiration  may 
be  gained  from  the  following  experiment.  A  number  of  similar  joints  were 
taken  from  the  same  plant  in  July  and  divided  into  three  lots. 

TABLE  26. 


Condition!. 

CO,  in  rag. 
per  gram 
fresh  weight 
per  hour. 

A  was  left  in  the  dark  for  2  days  at  the  air  temperatures  (July)  and 
the  CO2  emission  determined  at  28.°  

0.075 

B  was  left  in  the  dark  for  2  days  at  28°  and  the  C0a  emission  determined 
at  40  °  

0.034 

C  was  left  in  the  dark  for  2  days  at  12°  and  the  CO,  emission  determined 
at  28.°  

0.160 

It  is  evident  that  B,  which,  as  the  following  analysis  shows,  had  increased 
in  both  hexose  and  disaccharide  content  even  at  28°,  evolved  considerably 

1  DAVIS,  W.  A.    The  distribution  of  maltase  in  plants.    Exp.  Sta.  Record,  35,  413, 1916. 


62 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


less  CO2  at  40°  than  did  A  at  28°,  which  had  been  exposed  to  air-tempera- 
tures. C,  on  the  other  hand,  evolved  a  relatively  large  amount  of  CO2  at 
28°  after  having  been  kept  at  12°.  Richards1  found  that  the  maximum 
carbon  dioxid  production  for  Opuntia  versicolor  was  quite  definitely  at 
45°.  The  analyses  are  given  in  table  27. 

TABLE  27.  —  Analysis  of  joints  of  Opuntia  phceacantha  which  had  been  exposed  to 
different  temperatures.   Values  in  percentages  of  the  dry  material. 


A,    out- 
of-door 
temper- 
ature 
in  July. 

B,  28" 

C,  It.' 

A,   out- 
of-door 
temper- 
ature 
In  July. 

B,  28." 

0,  12.  • 

Water  

63  62 

60  98 

60  00 

Hex  oses  plusdisac- 

Total  sugars  . 

20  03 

24  50 

20  19 

0  80 

1  12 

1  28 

Total  polysaccha- 

Disaccharides  

0.18 

0.37 

0.59 

rides 

19  35 

23  30 

18  89 

0  62 

0  75 

0  69 

Total  hexose  sugars.  . 
Hexose  polysac- 

10.45 

11.47 

8.46 

Total  pentoses  
Pentosans  

9.26 
9.04 

12.32 
12.16 

11.07 
10.96 

charides  

9.72 

10.45 

7.28 

Pentoses  

0.82 

0.91 

0.80 

The  results  from  another  series  are  given  in  table  28. 

Here  the  plants  contained  a  relatively  large  amount  of  monosaccharides 
and  disaccharides  to  begin  with.  There  were  three  sets  of  Opuntia  joints : 
One  was  analyzed  immediately  and  represents  the  condition  of  the  joints  at 
the  end  of  the  arid  autumn,  when  the  low  temperatures  had  already  affected 
the  carbohydrate  content,  but  before  the  winter  rains  caused  a  further 
decided  increase  in  the  simpler  sugars.  Of  the  two  other  sets  of  joints  one 
was  kept  for  35  days  at  28°  in  the  dark  and  the  other  for  the  same  length 
of  time  at  12°.  The  analyses  are  given  in  table  28. 

TABLE  28. — Analysis  of  joints  of  Opuntia  phceacantha,  original  conditon  and  after 
being  kept  for  35  days  at  28°  and  12°,  in  percentages  of  the  dry  material. 


Origi- 
nal 
condi- 
tion. 

*#." 

*£•* 

Origi- 
nal 
condi- 
tion. 

Kept  at 
28°. 

KiT.at 

Total  loss  in  weight. 
Mg.  CO2  per  hour  at 
28°  per  gram  fresh 
weight 

0.091 

0.308 
69.90 
18.95 

16.73 

18.70 
0.056 

0.156 
67.00 
17.34 

16.00 

18.40 
0.098 

0.294 
66.40 
18.46 

16.63 

Total  hexose  sugars  . 
Hexoae  polysaccha- 
rides  

7.90 
6.04 

2.02 
1.04 
0.98 
10.45 
0.35 
10.10 

6.62 
5.49 

1.21 
0.73 
0.48 
10.13 
0.22 
9.91 

7.11 

5.47 

1.77 
0.96 
0.81 
10.74 
0.20 
10.54 

Hexoses    plus    disac- 

Mg.  CO2  per  hour  at 
28°  per  gram  dry 

Hexoses  . 

Water 

Total  sugars  

Pentoses  

Total  polysaccha- 
rides  

Pentosans  

The  proportional  values  of  the  various  groups  of  carbohydrates  are  given 
in  table  29. 


RICHABDS,  H.  M.    Acidity  and  gas  interchange  in  cacti.    Carnegie  Inst  Wash.  Pub. 
No.  209,  p.  49,  1915. 


EFFECT  OF  TEMPERATURE  ON  THE  CARBOHYDRATE-CONTENT.  63 


From  these  results  it  becomes  evident  that  at  higher  temperatures  the 
plant  tends  to  the  formation  of  polysaccharides,  while  at  lower  temperatures 
a  condition  of  general  inversion  seems  to  prevail. 


TABLE  29. 


Original 
condition. 

Kept  at  28". 

Kept  at  12*. 

Total  polysaccharides 

0  883 

0  913 

0  900 

Total  sugars 
Hexoses 

0  162 

0  088 

0  147 

Hexose  polysaccharides 
Monosaccharides 

0  080 

0  044 

0  061 

Total  polysaccharides 
Hexoses  plus  disaccharides 

0  107 

0  069 

0  096 

Total  sugars 

A  similar  modification  of  the  carbohydrates  concomitant  with  changes  in 
temperature  has  been  observed  in  a  variety  of  other  plants.  Lidforss1 
showed  that  all  evergreen  leaves  in  temperate  latitudes  are  quite  starch-free 
from  the  beginning  of  December  and  throughout  the  winter.  With  the 
advent  of  higher  temperatures  in  spring  starch  again  appears  in  the  chloro- 
plasts.  Similar  observations  have  been  made  by  Mer/  Haberlandt,*  and 
Schultz.4  That  temperature  is  here  actually  the  determining  factor  is 
demonstrated  by  the  fact  that  when  the  leaves  during  any  time  in  the  winter 
are  brought  to  a  higher  temperature,  starch  formation  takes  place  very 
readily.  Maximow "  has  made  similar  observations  and  has  called  atten- 
tion to  the  high  content  of  glucose  in  evergreen  leaves  during  the  winter. 
The  low  eutectic  point  of  glucose  solutions  is  regarded  as  a  protection 
against  freezing.  Miyake '  reports  similar  conditions  existing  in  Japan. 
In  the  winter  many  evergreen  leaves  in  the  middle  and  southern  parts  of 
Japan  contain  more  or  less  starch,  while  in  the  colder  parts  the  leaves  are 
found  to  be  starch-free.  The  very  interesting  experiments  of  Czapek T  bear 
directly  upon  the  question  under  discussion.  He  showed  that  at  low  tem- 
peratures the  sugar  concentration  in  the  cell  must  be  very  much  higher  in 
order  that  starch  formation  may  take  place  than  at  higher  temperatures. 

1  LIDFOBSS,  B.    Zur  Physiologic  und  Biologie  der  wintergruenen  Flora.    Bot  Central- 

blatt,  68,  33-44,  1896. 
1  MEB,  F.    De  la  constitution  et  des  fonctions  des  feuilles  hivernales.    Bull.  Soc.  BoL 

France,  23,  231,  1876. 
1  HABEBLANDT,  G.    Vergleichende  Anatomie  des  assimilatorischen  gewebesytems  der 

Pflanzen.    Jahrb.  f.  wiss.  Bot.,  13,  74,  1882. 
*  SCHULTZ,  E.    Ueber  die  Reservestoffe  in  immergruenen  Blaettern.    Flora,  71,  223, 

1888. 
•MAXIMOW,  N.  A.    Chemische  Schutzmittel  der  Pflanzen  gegen  Erfrieren.    Ber.  d. 

deut.  bot.  Ges.,  30,  52-65,  293-305,  504-516, 1912. 
'  MIYAKE,  K.    On  the  starch  of  evergreen  leaves  and  its  relation  to  photosynthesis 

during  the  winter.    Bot.  Gaz.,  33,  321-340,  1902. 
7  CZAPEK,  F.    Der  Kohlenhydrat-Stoffwechsell  der  Laubblaetter  im  Winter.    Ber.  d. 

deut.  bot.  Ges.,  19,  120-127,  1911. 


64  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

In  none  of  the  leaves  employed  by  Czapek  did  starch  formation  occur  at 
low  temperatures  with  sugar  concentrations  under  7  per  cent,  while  at 
ordinary  temperatures  a  1  per  cent  saccharose  solution  produced  starch  in 
abundance. 

All  of  these  results  indicate  that  the  enzymatic  equilibria  are  so  adjusted 
that  at  high  temperatures  there  takes  place  a  general  reversion  of  the  carbo- 
hydrates to  the  polysaccharides,  while  at  lower  temperatures  there  is  general 
inversion  to  the  simpler  sugars. 


VIII.  AEROBIC  AND  ANAEROBIC  RESPIRATION. 

Richards1  made  a  number  of  determinations  of  the  CO2  emission  and  the 
CO2/O2  ratio  of  Opuntia  versicolor  in  atmospheres  of  hydrogen  and  of 
nitrogen.  The  most  noteworthy  result  of  these  experiments  is  the  relatively 
high  rate  of  carbon-dioxid  emission  despite  the  absence  of  oxygen.  The 
amount  of  carbon  dioxid  produced  in  normal  air  at  21°  was  about  twice  that 
emitted  in  the  absence  of  oxygen,  while  at  30°  the  amounts  were  almost 
equal.  Thus  the  CO2  per  gram-hour  at  21°  was  in  air  0.24  and  0.25  mg., 
in  nitrogen  0.17  and  0.15  mg.,  while  at  35°  it  was  in  air  0.30  and  0.33  mg., 
and  in  nitrogen  0.38  mg.  The  acidity  as  determined  by  titration  does  not 
diminish  at  the  rate  at  which  it  does  in  the  presence  of  oxygen.  It  appears, 
therefore,  that  the  carbon  dioxid  which  is  produced  does  not  arise  from  the 
breaking  down  of  the  organic  acids  to  the  same  extent  as  in  the  normal 
cases.  Some  knowledge  of  the  role  of  the  carbohydrates  under  anaerobic 
conditions  seemed,  therefore,  highly  desirable. 

Joints  of  Opuntia  phceacanilia  were  selected  in  the  manner  previously 
described.  One  set  of  joints  was  analyzed  immediately,  another  set  was 
placed  under  a  large  bell-jar  provided  with  tubes  for  circulation  of  air  in  a 
dark  room  kept  at  20°,  and  a  third  set  was  placed  in  the  same  dark  room 
under  a  bell- jar  in  an  atmosphere  free  of  oxygen. 

Anaerobic  conditions  were  produced  by  the  employment  of  the  methods 
of  Ruzieka.*  The  bell-jar  (15  liters  capacity)  was  placed  in  a  shallow  pan 
containing  500  c,  c.  of  a  1  per  cent  aqueous  phenol  solution,  70  c.  c.  20  per 
cent  aqueous  Na2CO3  solution  and  50  grams  dextrose  to  which  was  added 
about  500  c.  c.  of  paraffin  oil.  This  mixture  served  as  a  seal.  In  this  pan 
stood  a  large  glass  triangle  on  which  was  placed  a  shallow  glass  dish  con- 
taining a  quantitiy  of  pyrogallol  and  a  stick  of  KOH.  In  this  dish  stood 
another  glass  triangle  which  supported  a  container  made  of  wire  screen  in 
which  had  been  placed  the  cactus  joints.  In  the  pyrogallol  dish  there  was 
also  placed  a  small  beaker  containing  an  indicator  solution  made  of  50  c.  c. 
1  per  cent  phenol  solution,  5  c.  c.  20  per  cent  Na2CO3  and  1  g.  c.  p.  dex- 
trose, to  which  was  added  0.5  c.  c.  prepared  sulphuric  acid  indigo  solu- 
tion. The  oxygen  was  removed  from  the  bell-jar  by  burning  a  small  jet  of 

1  RICHARDS,  H.  M.    L.  c.,  pp.  57,  84. 

1  ABDEBHAIJ>EN,  E.    Handbuch  der  Biochem.    Arbeitsmethoden.    Vol.  Ill  (2),  1239, 
1910. 


AEROBIC  AND  ANAEROBIC  RESPIRATION. 


65 


hydrogen  therein.  The  hydrogen  was  prepared  from  c.  p.  zinc  and  sul- 
phuric acid  and  was  washed  through  a  solution  of  10  per  cent  lead  nitrate 
and  one  of  10  per  cent  silver  nitrate.  The  oxygen  in  the  bell- jar  was  con- 
sumed slowly,  and  when  the  flame  went  out  the  hydrogen  was  immediately 
turned  off.  By  means  of  a  bent  glass  tube  and  a  burette,  oxygen-free  water 
was  added  then  to  the  dish  containing  pyrogallol  and  KOH.  Thus  the  last 
traces  of  oxygen  were  removed  from  the  atmosphere  in  the  bell-jar.  In  all 
probability  the  cactus  joints  contained  some  oxygen,  but  from  all  indica- 
tions this  was  soon  used  up. 

The  plants  were  allowed  to  remain  thus  for  7  days,  when  they  were 
analyzed  in  the  usual  manner.    The  results  are  given  in  table  30. 

TABLE  30. — Carbohydrate-content  of  Opuntia  phceacantha  in  original  condition  and 
after  having  been  kept  in  air  and  in  an  atmosphere  free  of  oxygen  for  7  days. 
Values  in  percentages  of  the  dry  material. 


Original 
condition. 

s| 
S« 

,j 

Original 
condition. 

Oxygen-free 
atmosphere. 

« 

Water  

80.00 
26.15 
19.20 
13.95 
7.70 

80.15 
21.60 
16.15 
10.25 
5.44 

79.80 
22.80 
17.38 
11.96 
7.33 

0.95 
5.92 
11.55 
10.87 
0.68 

0.66 
4.66 
10.73 
10.11 
0.62 

0.25 
4.88 
10.25 
9.41 
0.76 

Total  sugars. 

Total  polysaccharides.  .  . 
Total  hexose  sugars  
Hexose  polysaccharides  . 

Total  pentoses  

Pentosans  

Pentoses  .         

It  is  evident  that  under  anaerobic  as  well  as  aerobic  conditions,  consider- 
able amounts  of  carbohydrates  are  consumed.  This  fact  was  established 
long  ago  by  Lechartier  and  Bellamy1  for  fruits.  It  is,  however,  noteworthy 
that  the  plants  under  anaerobic  conditions  consumed  as  much  or  even 
slightly  more  carbohydrate  material  than  those  kept  in  air.  The  conclusion, 
therefore,  seems  justified  that  the  CO2  emission,  under  anaerobic  conditions, 
previously  referred  to,  was  the  result  of  carbohydrate  respiration. 

A  similar  experiment  was  carried  out,  using  Opuntia  versicolor.  The 
same  method  and  procedure  were  followed  as  in  the  preceding  experiment 
The  plants  remained  under  the  experimental  conditions  for  five  days.  The 
results  are  given  in  table  31. 

TABLE  31. — Carbohydrate-content  of  Opuntia  versicolor  in  original  conditions  and 
after  having  been  kept  in  air  and  in  an  atmosphere  free  of  oxygen  for  5  days. 
Values  in  percentages  of  the  dry  material. 


• 

Original 
condition. 

Oxygen-  free 
atmosphere. 

3 

1  Original 
condition. 

Si 
|J 

MM 
O 

i 

Water  

70.00 
18.90 
16.22 
12.85 
10.67 

70.80 
12.41 
11.65 
10.92 
10.42 

67.15 
15.15 
13.90 
10.03 
9.12 

0.61 
1.76 
5.72 
5.24 
0.49 

0.10 
0.55 
1.41 
1.16 
0.25 

0.28 
0.72 
4.83 
4.53 
0.30 

Total  polysaccharides.  .  . 
Total  hexose  sugars  
Hexose  polysaccharides. 

Total  pentose  sugars  

Pentoses  

1  LECHABTIEB,  G.,  and  F.  BELLAMY. 
466,  1809. 


De  la  fermentation  des  fruits.    Compt.  rend.,  69, 


66  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

Opuntia  versicolor  shows  the  same  behavior  as  Opuntia  phwacantha  in 
that  for  at  least  5  days  these  plants  can  live  with  no  apparent  untoward 
effects  in  an  atmosphere  containing  no  oxygen.  The  rate  of  carbohydrate 
consumption  is  higher  under  anaerobic  conditions  than  during  aerobic 
respiration.  This  is  probably  due  to  the  higher  energy  release  and  hence 
greater  efficiency  of  the  aerobic  process,  as  was  pointed  out  in  the  intro- 
ductory discussion.  The  water  loss  under  the  experimental  conditions  was 
naturally  low ;  the  plants  which  had  consumed  the  larger  amounts  of  carbo- 
hydrate showed  a  higher  percentage  of  water-content. 

In  the  foregoing  experiment  with  Opuntia  versicolor  acidity  determina- 
tions were  also  made.  In  each  case  100  grams  of  the  plant  material  which 
had  been  ground  were  thoroughly  expressed  in  a  meat-press.  The  juice 
thus  obtained  was  made  up  to  a  definite  volume  and  aliquot  portions  titrated 
with  0.1  normal  potassium  hydrate,  using  phenolphthalein  as  an  indicator. 
The  plants  representing  the  original  condition  were  collected  at  4  p.  m.,  at  a 
time  when  the  acidity  was  almost  at  its  minimum.  The  results  thus  obtained 
are  as  follows : 

Original  condition 0.20  c.  c.  0.1  normal  ROH  per  gram  fresh  material 

Kept  in  air 0.78  c.  c.    " 

In  oxygen-free  atmosphere 0.50  c.  c.   "        "  "       " 

From  this  it  is  evident  that  acid  formation  is  benefited  by  the  presence  of 
oxygen.  This  is  quite  in  accord  with  the  results  obtained  by  Warburg.1 
The  principles  underlying  the  phenomena  of  acidification  and  deacidifica- 
tion  have  been  discussed  by  this  worker  and  more  recently  by  Eichards  and 
by  the  author/  It  is  evident  also  that  there  is  a  decided  acidification  even 
in  the  absence  of  oxygen.  Whether  this  acidity  also  represents  malic  acid, 
as  under  normal  conditions,  or  some  other  acid,  the  product  of  real  intra- 
molecular respiration  is  an  open  question.  The  answer  hereto  would  throw 
considerable  light  on  the  mode  of  this  form  of  energy  release. 

The  respiration  of  succulent  plants  represents  somewhat  of  a  modifica- 
tion of  the  process  taking  place  in  most  mesophytes.  The  characteristic 
formation  of  acids  is  intimately  associated  with  the  restricted  oxygen  supply 
consequent  on  the  structure  of  the  succulent  type.  However,  when  these 
plants  are  deprived  of  all  oxygen,  it  is  evident  that  the  final  course  which 
the  respiratory  process  follows  is  further  altered,  and  the  products  formed 
are  radically  different.  In  consideration  simply  of  the  chemical  reactions 
of  sugars  in  aqueous  solution  in  the  presence  of  oxidizing  agents  under 
various  conditions,  as  outlined  in  the  introductory  discussion,  variations  in 
the  reaction  products  with  altered  external  conditions  of  the  organism  are  to 
be  expected.  It  was  indicated  in  this  discussion  in  what  manifold  channels 
the  course  of  sugar  disintegration  may  proceed,  and  what  slight  differences 
in  the  nature  of  solution  may  affect  the  main  course  of  the  reaction.  Pre- 
cursory to  oxidation  of  the  sugars,  there  must  take  place  a  dissociation  or 

*WABBUBG,  O.  Ueber  die  Bedeutung  der  Organischen  Saeuren  fuer  den  Lebenspro- 
zess  der  Pflanzen.  Speciell  der  Sag.  Fettpflanzen.  Unters.  aus  dem  Bot. 
Inst.  zu  Tuebingen,  2,  53-150,  1886. 

1  SPOEHB,  H.  A.    Biochem.  Zeitschr.,  57,  95, 1913. 


AEROBIC  AND  ANAEROBIC  RESPIRATION.  67 

splitting  of  the  sugar  molecule.  Such  an  assumption  finds  physiological 
substantiation  in  the  fact  established  by  Bertrand,1  that  oxidases  are  not 
capable  of  directly  oxidizing  carbohydrates.  The  extensive  researches  of 
Godlewski  and  of  the  Russian  school  of  Palladin  and  Kostytschew  have 
made  notable  experimental  contributions  to  a  clearer  understanding  of  this 
subject. 

It  is  still  a  matter  of  dispute  as  to  what  are  the  agencies  which  effect  this 
disruption  of  the  sugar  molecule.  By  some  it  has  been  maintained  that  all 
respiration  has  as  its  beginning  zymase  fermentation,  and  that  the  products 
of  this  action  are  then  oxidized  to  form  the  familiar  products  of  aerobic 
respiration.  However,  the  results  of  Kostytschew2  and  others  make  it 
appear  highly  improbable  that  the  higher  plants  oxidize  alcohol  formed 
either  as  an  intermediate  product  or  when  given  the  plant  as  nutrient 
Kostytschew  and  Palladin1  also  showed  that  some  plants  exhibit  a  mixed 
type  of  anaerobiosis  in  which  the  nature  of  sugar  disintegration  is  different 
from  the  regular  zymase  fermentation.  In  potatoes  the  amount  of  alcohol 
formed  was  exceedingly  small;  while  from  the  results  with  various  leaves 
they  conclude  that  about  one-half  of  the  CO2  production  is  the  result  of 
zymase  action,  the  rest  of  the  CO2  resulting  from  a  different  form  of  gly- 
colysis.  It  is  to  be  concluded,  .then,  that  in  the  plant  sugar  disintegration 
may  follow  several  courses  concomitantly,  and  that  the  nature  of  the  main 
metabolic  product  depends  upon  which  of  these  courses  is  favored.  This  is, 
of  course,  just  what  would  be  expected  from  a  consideration  of  the  behavior 
of  sugar  solutions  under  the  influence  of  various  catalytic  agents  as  revealed 
by  the  extensive  investigations  of  Nef,  which  have  been  already  discussed. 

It  seemed,  therefore,  not  without  interest  to  determine  how  the  cacti, 
which  under  normal  conditions  exhibit  a  rather  modified  course  of  respira- 
tion, behaved  under  anaerobic  conditions.  The  procedure  consisted  essen- 
tially of  expressing  the  juice  from  200  grams  of  the  ground  cactus  and 
determining  the  alcohol  in  this  juice.4  A  small  portion  of  the  juice  was 
used  for  the  determination  of  acidity ;  the  remainder  was  used  for  the  alcohol 
determinations.  By  addition  of  calcium  carbonate,  the  juice  was  made 
neutral.  To  this  neutralized  juice  there  was  added  ammonium  sulphate, 
75  grams  of  the  salt  to  each  100  c.  c.  of  juice.  This  was  carried  out  in  a 
wide-mouth  bottle  and,  after  the  addition  of  the  ammonium  sulphate  and  a 

IBEBTKAND,  G.  Sur  les  rapports  qui  existent  enter  la  constitution  chemique  des 
composes  organiques  et  leur  oxidabilite"  sous  1'influence  de  la  laccase.  Comp. 
rend.,  122,  1132,  1896. 

MATHEWS,  A.  P.    The  spontaneous  oxidation  of  sugars.    Jour.  Biol.  Chem.,  6,  1-20, 
1909. 

1  KOSTYTSCHEW,  S.  Ueber  den  Zusammenhang  der  Sauerstoffatmung  der  Samen- 
pflanzen  mit  der  Alkoholgaerung.  Ber.  d.  deut.  bot.  Ges.,  26,  565-573,  1908. 

8  PALLADIN,  W.,  and   S.  KOSTYTSCHEW.     Ueber  die  anaerobe  Atmung  der   Samen- 

pflanzen  ohne  alkohol  bildung.    Ber.  d.  deut.  bot.  Ges.,  25,  51-56,  1907. 
KOSTYTSCHEW,  S.    Ueber  das  Wesen  der  anaeroben  Atmung  verschiedener  Samen- 
pflanzen.    Ber.  d.  deut.  bot.  Ges.,  31,  125-129,  1913. 

4  Box,  ARTHUR  W.,  and  A.  R.  LAMB.  An  accurate  aeration  method  for  the  determina- 
tion of  alcohol  in  fermentation  mixtures.  Jour.  Amer.  Chem.  Soc.,  38,  2561- 
2568,  1916. 


68 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


few  drops  of  toluene,  the  bottle  was  immediately  attached  to  the  aeration 
train.  The  alcohol  was  removed  from  the  juice  by  forcing  a  stream  of  air 
through  the  solution  and  absorbing  the  alcohol  which  is  thus  distilled  over 
in  concentrated  sulphuric  acid. 

The  arrangement  was  as  follows :  The  air  passed  first  through  a  solution 
of  sulphuric  acid  and  potassium  bichromate,  then  through  a  safety  bottle, 
whereupon  it  was  forced  through  the  plant  juice,  another  small  safety  bottle, 
and  then  through  two  small  absorbing  cylinders  containing  concentrated 
sulphuric  acid.  The  air  was  passed  through  this  train  for  24  hours.  The 
sulphuric  acid  was  then  transferred  to  a  distilling  flask  connected  with  a 
condenser  and  receiving  vessel.  By  means  of  a  dropping-funnel  a  2  per 
cent  solution  of  potassium  bichromate  was  added  to  the  sulphuric  acid. 
The  warm  mixture  in  the  distilling  flask  was  allowed  to  stand  for  15  to  20 
minutes  in  order  to  complete  the  oxidation  of  the  alcohol  to  acetic  acid,  and 
then  distilled  until  no  more  acid  came  over.  A  thermometer  was  kept  in  the 
solution  and  the  temperature  was  not  allowed  to  rise  over  112°.  It  was 
found  that  at  higher  temperature  there  is  danger  of  sulphuric  acid  being 
carried  over.  The  solution  can  be  distilled  until  the  distillate  is  neutral  to 
litmus  by  adding  25  c.  c.  of  boiled  water  from  time  to  time  to  prevent  the 
solution  from  becoming  too  concentrated.  The  distillate  was  made  up  to  a 
definite  volume  and  titrated  with  0.1  normal  potassium  hydroxide. 

As  has  been  stated,  the  nocturnal  respiration  of  the  cacti  is  characterized 
by  the  formation  of  acids.  This  acidification  is  the  result  of  restricted 
oxygen  supply.  The  question  arises,  then,  whether  under  these  conditions 
there  is  also  formation  of  alcohol — that  is,  whether  the  course  of  respiration 
is  so  affected  as  to  radically  change  the  mode  of  the  process.  For  this 
purpose  Opuntia  versicolor  joints  were  collected  in  the  morning  and  in  the 
evening,  and  acidity  and  alcohol  determinations  made  in  the  usual  manner. 
The  results  are  given  in  table  32. 

TABLE 32. — Diurnal  variations  in  the  acidity  and  alcohol  content  of  Opuntia  versicolor, 

May  7-8. 


Collected  at 
4»30«  p.  m. 

Collected  at 
8»15"*  a.  m. 

Water-content  

75  67 

75  80 

Acidity  in  cubic  centimeters  0.1  N  KOH  per  100  grams 
fresh  material  

20  30 

37  65 

Alcohol  in  grams  per  100  grams  of  fresh  material  

0.0066 

0.0032 

There  is  evidently  no  accumulation  of  alcohol  in  these  plants  during  the 
night  time  coincident  with  the  nocturnal  acidification.  It  seems  highly 
improbable  that  this  form  of  respiration  can  be  regarded  as  being  of  intra- 
molecular nature.  It  was  observed,  however,  that  there  was  a  distinct 
increase  in  the  alcohol-content  of  these  plants  after  they  had  been  exposed 
to  sunlight  for  some  time.  This  fact  serves  to  substantiate  the  theory  of 


AEROBIC  AND  ANAEROBIC  RESPIRATION. 


69 


acid  disintegration  which  was  advanced  by  the  writer  in  an  earlier  paper/ 
The  acidity  of  these  cacti  in  the  morning  is  due  essentially  to  the  presence 
of  malic  acid.  The  photolysis  of  malic  acids  yields  through  the  loss  of  two 
molecules  of  carbon  dioxid  from  the  carboxyl  groups  one  molecule  of  ethyl 
alcohol. 


COOH 
CH2 
CHOH 
COOH 


CH3 

2COa+| 

CHa 


OH 


As  the  photolytic  disintegration  of  the  malic  acid  proceeds  there  is  thus 
an  accumulation  of  ethyl  alcohol. 

Under  anaerobic  conditions  there  is  a  very  active  production  of  alcohol 
in  the  cacti.     In  table  33  are  given  the  results  of  analyses  of  plants  of 

TABLE  33. — Acidity  and  alcohol  content  of  Opuntia  versicolor  after 
being  kept  in  air  and  oxygen-free  atmosphere. 


Atmosphere. 

Time  in 
hours. 

Water- 
content. 

Acidity  in  c.c. 
0.1  N  KOH  per 

100  grama  fresh 
material. 

Alcohol  in 
grams  per  100 
grama  fresh 
material. 

Air  

168 

74.30 

83.57 

0.0041 

O.-f  ree                    

192 

78.40 

47.40 

0.3800 

Air  

168 

73.40 

86.80 

0.0040 

02-f  ree  

144 

77.05 

67.80 

0.0189 

Opuntia  versicolor  which  were  kept  in  an  atmosphere  freed  of  oxygen  in  the 
manner  previously  described. 

The  normal  amount  of  alcohol  is  very  small  in  Opuntia  versicolor.  In 
the  course  of  normal  respiration  in  the  dark  this  quantity  does  not  increase, 
although  the  acidity  rises  very  much.  When  kept  in  an  atmosphere  freed 
of  oxygen  large  quantities  of  alcohol  are  formed;  there  is  also  some  acid 
formation,  but  this  is  not  nearly  so  great  as  in  air.  It  is  very  noticeable 
that  due  to  the  higher  rate  of  sugar  consumption  under  anaerobic  condi- 
tions, the  water-content  of  plants  kept  under  such  conditions  is  finally  much 
higher  than  in  those  kept  in  air. 

*  SPOEHB,  H.  A.    Blochem.  Zeitsch.,  57, 106, 1913. 


70  THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 

IX.    CONSUMPTION   OF   CARBOHYDRATES   DURING 
STARVATION. 

A  very  striking  property  of  many  of  the  cacti  is  their  ability  to  survive 
long  periods  of  drought  and  starvation.  A  notable  record  of  this  has  been 
made  by  MacDougal  et  al.1  on  the  desiccation  of  Echinocactus.  This  plant, 
left  in  the  diffuse  light  of  a  room  without  being  given  any  water,  after  six 
years  had  the  same  proportion  of  water  as  at  the  beginning,  although  it  had 
lost  almost  30  per  cent  of  its  original  weight.  It  has  since  been  found  that 
similar  conditions  obtain  in  many  other  cacti.  Here  are  given  the  records 
of  the  water-loss  and  rate  of  carbohydrate  depletion  of  two  sets  of  Opuntia 
phceacantha.  For  each  series  a  large  number  of  joints  of  the  same  age  were 
taken  from  one  plant  and  placed  in  a  dark  room,  the  temperature  of  which 
was  maintained  throughout  the  experiment  at  28°  C.  The  joints  were 
placed  upon  racks  permitting  free  circulation  of  air.  For  over  a  year  they 
remained  in  apparently  perfectly  healthy  condition.  At  the  end  of  this 
time  a  number  of  the  joints  were  planted  in  soil  and  watered,  within  a  week 
they  put  out  roots,  and  shortly  thereafter  developed  vegetative  buds. 

If  a  rather  definite  water-content  is  to  be  maintained  during  long 
periods  while  a  plant  is  actually  losing  large  quantities  of  water  there 
must,  of  course,  be  a  simultaneous  loss  of  dry  material  through  respira- 
tion. As  has  been  shown,  this  dry  material  is  essentially  of  carbohydrate 
nature.  A  considerable  portion  thereof  is  in  the  form  of  cellulose,  however, 
and  from  the  investigations  by  MacDougal  et  al.  it  is  evident  that  this 
cellulose  may  be  utilized  as  food  material  by  the  plant  under  circumstances 
of  stress.  Added  to  the  tendency  of  the  plant  to  maintain  its  water-balance 
by  a  concomitant  loss  of  dry  material  is  the  factor  that  the  burning  of  sugar 
also  results  in  the  formation  of  water.  For  each  gram  of  sugar,  calculated 
as  C6Hi2O6,  there  is  formed  0.6  gram  of  water.  When  the  water-content 
TABU:  34.  of  the  plant  is  already  high  this  factor  is,  of  course, 

quite  insignificant,  but  when  the  water-content  is 
low  the  water  added  thereto  by  the  burning  of  sugar 
may  be  of  some  significance  in  the  economy  of  the 
plant.  Naturally,  the  maintenance  of  the  water- 
balance  is  entirely  a  relative  matter,  the  rates  at 
which  respiration  and  transpiration  may  proceed 
and  the  plant  still  maintain  its  water-balance  de- 
pend upon  the  original  condition.  Thus,  in  order  to  maintain  its  original 
water-balance  and  retaining  a  constant  rate  of  respiration,  the  plant  may 
lose  the  following  quantities  of  water  at  the  water-content  indicated  in  the 
accompanying  table.  For  each  gram  of  dry  material,  calculated  as  sugar, 
lost  by  respiration  the  water-loss  equaled  as  shown  in  table  34. 

*  MAcDotTQAJU  D.  T.,  E.  R.  LONG,  and  J.  G.  BROWN.    End  results  of  desiccation  and 
respiration  in  succulent  plants.    Physiological  Researches,  1,  289-325,  1915. 


At  water 
content  of — 


f.ct. 
60 
75 
80 


Water  loss. 


gram*. 
2.1 
3.5 
4.5 
9.6 


CONSUMPTION   OF  CARBOHYDRATES  DURING  STARVATION.     71 


In  order  that  the  water-balance  be  maintained,  the  lower  the  water- 
content  the  less  can  be  the  rate  of  water-loss  at  a  constant  rate  of  respiration ; 
and,  conversely,  the  lower  the  water-content  the  higher  can  be  the  respira- 
tion at  a  constant  rate  of  transpiration.  The  examples  given  in  tables  35 
and  36  will  illustrate  the  point. 

TABLE  35. — Loss  in  weight  and  rate  of  C0t  emission  of 
Opuntia  phccacantha  kept  in  dark  at  28°  C. 


Day.. 

Original 
weight. 

Loss, 
grama. 

per  cent. 

Mg.  CO,  per 
hour  per 
gram  dry 

Water- 
content. 

weight. 

0  724 

84  75 

33 

426 

83.0 

19.40 

0.247 

83.75 

55 

474 

100.0 

21.09 

0.249 

84.50 

91 

546 

167.5 

30.60 

0.185 

81.76 

118 

443 

271.5 

61.20 

0.090 

72.68 

150 

465 

280.0 

60.20 

0.095 

75.32 

174 

565 

240.0 

42.50 

0.128 

80.00 

189 

480 

253.0 

52.50 

0.081 

75.48 

The  plants  in  table  35  had  a  relatively  high  initial  water-content;  they 
were  kept  at  28°  with  a  constant  movement  of  air  and,  although  the  losses 
were  not  uniform,  they  lost  over  60  per  cent  of  their  total  weight ;  the  water- 
content  was  reduced  by  but  12  per  cent  The  plants  in  table  35  started  with 
a  much  lower  water-content,  which  is  reduced  by  only  9  per  cent,  with 
decidedly  lower  total  loss  of  weight  and,  relative  to  the  total  loss,  a  higher 
rate  of  respiration. 

TABLE  36. — Loss  in  weight  and  rate  of  C0t  emission  of 
Opuntia  phosacantha  kept  in  dark  at  28°  C. 


Days. 

Original 
weight. 

Loss, 
grams. 

Los., 
per  cent. 

Mg.  CO,  per 
hour  per 
gram  dry 

Water- 
content. 

weight. 

169.5 

0.250 

68.50 

54 

203.0 

48 

23.6 

0.130 

63.20 

72 

186.0 

39 

21.0 

0.094 

63.80 

92 

183.0 

55 

27.3 

0.147 

59.87 

121 

201.0 

66 

32.9 

0.074 

59.50 

143 

209.0 

67 

32.1 

0.052 

62.40 

One  example  will  suffice  to  illustrate  the  manner  in  which  the  two  factors 
of  respiration  and  transpiration  work  together  to  maintain  the  water- 
balance.  In  table  36  the  first  and  second  sets  may  be  taken.  In  the  initial 
condition  the  plants  emitted  0.25  mg.  CO2  per  hour  per  gram  dry  material, 
i.  e.,  they  were  consuming  at  the  rate  of  0.00017  gram  of  sugar  (C6HuO«) 
per  hour  per  gram  of  dry  material,  or,  calculated  for  the  second  set  of  plants, 
0.0108  gram  of  sugar  per  hour.  After  54  days  there  was  produced  CO2 
at  the  rate  of  0.13  mg.  per  hour  per  gram  of  dry  material,  equal  to  0.000089 
gram  of  sugar  per  hour  per  gram  dry  material,  or  0.0051  gram  sugar  per 
hour  for  the  entire  set  of  plants.  This  would  average  0.0079  gram  of  sugar 


72 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


used  per  hour  by  the  plants  or  10.24  grams  of  sugar  for  the  54  days.  The 
original  63.90  grams  of  dry  material  of  the  plant  was  then  reduced  by  10.24 
grams,  leaving  53.66  grams.  The  burning  of  10.24  grams  of  sugar  yields 
6.14  grams  of  water,  or  results  in  a  net  loss  of  4.10  grams.  The  plants 
showed  a  total  loss  of  48.0  grams,  hence  this  quantity  less  4.10  grams  repre- 
sents the  amount  of  water  lost,  i.  e.,  43.90  grams.  There  were  present- 
originally  139  grams  of  water  in  the  plants;  less  43.90  grams  would  leave 
95.10  grams;  to  this  must  be  added  the  amount  of  water  produced  by  the 
oxidation  of  the  carbohydrates,  which  brings  the  water-content  to  101.24 
grams.  From  the  values  thus  calculated  (53.66  grams  of  dry  material  and 
101.24  grams  of  water)  there  results  a  theoretical  water-content  of  65.35  per 
cent.  The  value  actually  found  was  63.20  per  cent. 

There  are,  of  course,  several  factors  which  modify  the  calculated  results. 
Most  important  of  these  is  the  rate  of  carbon-dioxid  emission.     This  is 


\" 


FIG.  2. 


probably  too  high  and  would  result  in  bringing  the  calculated  water-content 
to  too  high  a  value.  As  can  be  seen  from  figure  2,  the  curve  of  the  rate  of 
carbon-dioxid  emission  does  not  decrease  in  a  straight  line,  but  rather 
abruptly,  so  that  the  total  amount  of  sugar  consumed  is  probably  calculated 
too  high.  The  abrupt  drop  in  the  curve  of  carbon-dioxid  emission  is  not  a 
traumatic  effect ;  the  cause  has,  however,  as  yet  not  been  discovered. 

It  is  evident  that  these  plants  do  not  go  into  a  condition  of  rest  or 
dormancy  when  the  water-supply  is  greatly  diminished,  but  continue  their 
respiratory  activity  at  about  the  same  rate  as  when  supplied  with  abundant 
water.  This  fact  has  already  been  pointed  out  by  Kichards.  It  would 
seem  that  this  is  made  possible  by  virtue  of  their  ability  to  use  as  food 
material  not  only  the  simpler  monosaccharides  which  under  these  conditions 
are  present  in  reduced  quantities,  but  also  the  polysaccharides.  How  this 
results  in  the  formation  of  pentosans  in  the  plant  will  be  discussed  in  a 
later  section. 


CONSUMPTION  OF  CARBOHYDRATES  DURING  STARVATION.     73 


The  course  of  the  depletion  of  the  stored  carbohydrates  can  be  seen  in 
table  37.  The  proportion  of  the  various  sugars  maintains  a  surprising 
regularity  as  the  depletion  proceeds.  This  is  an  important  fact  in  view  of 
the  opinion  which  has  been  frequently  expressed  regarding  the  physiological 
role  of  the  pentoses.  These  sugars  have  been  considered  by  some  plant 
physiologists  as  end  or  waste  products  of  the  normal  metabolism,  which 
accumulate  as  the  plant  becomes  older.  Were  this  the  case  it  would  be 
expected  that,  in  plants  subjected  to  conditions  such  as  these,  there  would  be 
an  increase  in  pentoses,  at  least  relative  to  the  other  sugars.  But  this  is 
found  not  to  be  the  case ;  the  pentoses  apparently  are  drawn  into  the  course 
of  carbohydrate  metabolism  to  a  marked  degree.  The  starch,  which  is 
present  in  very  numerous  minute  grains,  disappears  slowly.  After  72  days 
there  are  visible  but  a  few  scattered  grains ;  and  after  92  days  practically 
all  of  the  starch  has  disappeared. 

TABLE  37. — Carbohydrate-content  of  Opuntia  ph&acantha  during  starvation.    Values 
in  percentages  of  the  dry  material. 


Da 

?B. 

0 

54 

72 

92 

121 

143 

Water  

63.50 

63.20 

63.80 

59.87 

59.50 

62.40 

Total  sugars 

22  86 

16  62 

13  71 

15  25 

14  54 

13  52 

Total  polysaccharides  

20.38 

14.22 

12.30 

14.10 

13.52 

12.46 

Total  hexose  sugars  

19.74 

14.85 

12.25 

13.27 

12.24 

11.70 

Hexose  polysaccharides  

17.64 

13.72 

11.15 

12.41 

11.48 

10.87 

Hexoses  plus  disaccharides  

2.25 

1.21 

1.18 

0.91 

0.81 

0.89 

Disaccharides  

1  63 

0  72 

0.73 

0.70 

0.55 

0.59 

Hexoses  .         

0.62 

0.49 

0.45 

0.21 

0.26 

0.30 

Total  pentose  sugars              

2.93 

1.68 

1.38 

1.84 

2.17 

1.72 

2  67 

1.33 

1  08 

1.25 

2.02 

1.50 

0.36 

0.34 

0.30 

0.25 

0.15 

0.22 

0  98 

0  83 

0  75 

0  46 

0.31 

0.52 

Another  experiment  was  undertaken  to  determine  the  effect  of  starvation 
on  the  subsequent  photosynthetic  activity  as  well  as  of  feeding  with  cane- 
sugar  and  dextrose.  A  number  of  joints  of  Opuntia  phaacantha  were  left 
in  the  dark  at  28°,  as  previously  described,  for  189  days.  One  set  of  joints 
(A)  was  then  analyzed  to  determine  the  status  of  the  carbohydrates.  The 
remaining  joints  were  placed  in  battery  jars  containing  water,  in  such  a 
manner  that  the  cut  end  was  just  below  the  surface  of  the  water.  Within 
10  days  all  the  joints  had  produced  roots  2  to  5  cm.  in  length.  The  plants 
were  then  divided  into  3  lots  of  7  joints  each  and  treated  in  various  ways. 
B  was  placed  with  the  roots  in  a  sterile  1  per  cent  cane-sugar  solution ;  C 
with  roots  in  a  sterile  1  per  cent  dextrose  solution,  both  in  the  dark  at  28°  ; 
and  D  with  the  roots  in  a  nutrient  solution,  in  the  sunlight  The  plants 
remained  thus  for  60  days ;  the  solutions  were  changed  frequently.  At  the 
end  of  this  time  the  plants  were  all  in  a  very  healthy  condition  and  were 
analyzed  in  the  usual  manner;  the  results  are  given  in  table  38. 


74 


THE  CARBOHYDRATE  ECONOMY  OF   CACTI. 


The  gain  in  water-content  of  the  plants  which  had  been  placed  with 
their  roots  in  solutions  is  very  apparent.  The  effect  on  the  carbon-dioxid 
production  is  very  striking  in  the  case  of  the  cane-sugar  and  dextrose ;  both 
show  a  very  decided  increase.  The  plants  which  were  in  the  sunlight  show 
but  a  very  slight  gain.  In  none  of  the  cases  is  the  gain  in  carbohydrate- 
content  very  great  This  was  somewhat  surprising,  as  the  plants  before 
the  starvation  contained  20.60  per  cent  total  sugars,  4.50  per  cent  hexoses, 
and  8.28  per  cent  total  pentoses.  The  plants  in  a  solution  of  cane-sugar 

TABLE  38. — Carbohydrate-content  in  percentages  of  the  dry  material  of  Opuntia 
phceacantha. 


A 

B 

C 

D 

0  080 

0.777 

0  865 

0  116 

Water       

75.48 

84.55 

85.45 

88.90 

Total  sugars  

1].50 

13.00 

10.70 

12.00 

Total  polysaccharides  •.  

11.05 

12.15 

10.06 

11.78 

Total  hexose  sugars  

8.88 

11.02 

9.27 

10.62 

8.58 

10.44 

8.84 

10.34 

0.33 

0.63 

0  47 

0.30 

Disacchar  ides.              

0.21 

0.48 

0.28 

0.24 

0.12 

0.15 

0.19 

0.06 

2  49 

1.87 

1.40 

1.31 

Pentosans  

2.35 

1.62 

1.24 

1.09 

Pentoses.     .         

0.14 

0.25 

0.21 

0.22 

A  starved  189  days,  B  subsequently  for  60  days  in  1  per  cent  cane-sugar,  C  in  1  per 
cent  dextrose,  D  in 'sunlight. 

show  a  greater  gain  in  disaccharides,  while  the  plants  in  dextrose  show  a 
greater  gain  in  hexoses.  The  joints  which  had  been  in  the  sunlight  exhibited 
but  very  slight  gains,  the  hexoses  being  even  less  than  in  the  original 
starved  condition.  This  is  probably  of  considerable  importance  for  an 
understanding  of  the  nature  of  the  photosynthetic  process.  It  has  been 
found  that  a  similar  condition  exists  in  thin  leaves,  the  rate  of  photosyn- 
thesis varying  with  the  rate  of  respiration,  suggesting  an  intimate  associa- 
tion between  photosynthesis  and  the  respiratory  activity.  Investigation  on 
this  subject  is  now  in  progress.  In  all  three  cases  the  pentosans  show  the 
usual  diminution  with  increased  water-content. 


THE  ORIGIN  AND  ROLE  OF  PENTOSE  SUGARS.  75 

X.  THE  ORIGIN  AND  ROLE  OF  PENTOSE  SUGARS. 

The  pentose  sugars  in  plants  have  been  most  familiar  as  pentosans,  as 
components  of  the  cell  walls  and  vessels  of  the  plants,  and  as  found  in 
various  gums  in  the  form  of  xylan  and  araban.  While  widespread  in  the 
vegetable  kingdom,  the  pentose  sugars  have  but  recently  been  regarded  in 
their  important  bearing,  and,  in  fact,  were  for  a  long  time  not  recognized 
as  belonging  to  a  separate  group.  In  fact,  the  presence  of  monosaccharide 
pentoses  has  been  but  very  recently  established.  That  the  pentoses  are  of 
great  physiological  importance  to  the  plant  becomes  evident  in  the  light 
of  recent  investigations  on  the  chemistry  of  the  cell-nucleus.  Among  the 
chief  components  hereof  are  the  so-called  nucleic  acids. .  These  are  highly 
complex  substances  consisting  roughly  of  a  combination  of  phosphoric  acid, 
purines,  pyrimidines,  and  several  carbohydrate  groups.  The  plant  nucleic 
acids  so  far  studied  have  been  found  to  contain  the  pentose  group.1  The 
function  and  fundamental  importance  of  the  nucleus  in  the  metabolism  of 
the  plant  need  no  further  discussion  here ;  however,  a  more  intimate  knowl- 
edge of  the  chemical  composition  and  action  will  undoubtedly  lead  to  a 
clearer  understanding  of  the  intricate  reactions  of  this  most  important 
organ. 

It  is  evident,  then,  that  the  5  carbon  atom  group  of  sugars  is  a  common 
component  of  plants  and  is  of  great  importance  in  some  of  the  most  vital 
metabolic  activities  of  the  organism.  Nevertheless,  the  origin  and  mode  of 
formation  of  the  pentose  sugars  is  still  quite  obscure.  This  problem  is  of 
special  interest  because  any  light  thereon  would  be  of  great  value  in  gaining 
a  clearer  understanding  of  the  process  of  the  photosynthetic  appropriation 
of  carbon  dioxid  by  the  chlorophyllous  leaf.  The  question  resolves  itself 
into  whether  the  pentoses  are  direct  products  of  photosynthesis  or  are  derived 
from  other  sugars  through  subsequent  metabolic  activity.  If,  for  instance, 
the  formation  of  sugar  in  the  green  leaf  actually  takes  place  by  means  of  a 
progressive  addition  of  six  molecules  of  formaldehyde,  the  presence  of 
pentoses  is  to  be  expected. 

2CH2O->C2H4O2;  C2H4O2  -f  CH20->C,HaO, ; 

C8H6Os  +  CH2O-^C1H8O4 ;  C4H2O4  +  CB^O— ^C.H^OB  ; 

C5H10O6  +  CH20->C6H12O6. 

If,  again,  the  sugars  are  formed  by  the  union  of  two  molecules  of  gly- 
cerine aldehyde,  hexoses  or  their  condensation  products  would  be  the  only 
substances  formed: 

2CH2OH  •  CHOH  •  CH:  O,    CH2OH(CHOH)4  •  CH:  O. 

This,  however,  is  purely  chemical  speculation  and  so  far  has  aided  little 
in  the  solution  of  the  problem  of  photosynthesis,  the  real  course  of  which  is 
probably  far  more  complicated  than  has  been  generally  assumed.  Not 

1  LEVENE,  J.,  and  W.  JACOBS.    Ueber  die  Pankreas-Pentose.    Ber.  d.  deut  chem.  Get., 

43,  3147-3150,  1910. 
.    Ueber  die  Triticon  Nucleinsaeure.    Ibid.,  43,  3164-3167,  1810. 


76 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


enough  work  has  been  done  with  the  chlorophyllous  leaf  to  draw  valid  con- 
clusions, and  this  in  turn  has  been  due  to  the  fact  that  the  methods  of  sepa- 
ration and  determination  have  not  been  sufficiently  delicate  and  accurate  to 
permit  their  application  to  such  sensitive  material. 

Chalmont,1  Tollens,'  and  Windish  and  Hasse*  come  to  the  conclusion  that 
pentoses  are  formed  from  hexoses  as  first  products  of  oxidation,  that  they 
are  relatively  inert,  and  probably  are  of  the  nature  of  waste  products.  This 
is  based  essentially  upon  their  observations  that  the  total  pentosan-content  of 
seedlings,  germinated  and  grown  in  the  dark,  increases  with  age.  The 
writer  made  determinations  of  the  total  pentoses  in  seedlings;  the  results 
are  quite  the  reverse,  however.  Wheat  seeds  were  allowed  to  germinate  on 
glass  wool  in  the  dark;  at  intervals  a  number  of  seedlings  were  removed, 
dried,  ground,  hydrolyzed  with  1  per  cent  HC1,  and  after  determining  and 
fermenting  away  the  hexose  sugars  the  pentose  sugars  were  determined 
(see  table  39).4 

TABLE  39. — Total  hexose  and  pentose  content  of  wheat  seedlings. 


Days. 

0 

1 

3 

6 

8 

Dry  weight  

91.80 
43.35 
47.50 
7.60 
8.30 

63.20 
25.90 
41.00 
6.24 
3.95 

54.00 
25.10 
46.50 
1.75 
3.24 

34.80 
18.94 
54.50 
0.57 
1.66 

21.40 
9.95 
46.50 
0.26 
0.05 

Total  hexose  sugar  -1  J£r 

_  ,   ,                   f  fresh  .  . 

Total  pentoses  4  Y~~ 

Unquestionably  many  organisms  utilize  the  pentose  sugars  as  sources  of 
energy.  In  their  physiological  effects  the  differences  between  the  pentoses 
and  hexoses  are  in  many  cases  less  than  exist  between  closely  related  mem- 
bers of  either  one  of  the  groups.  Many  bacteria  and  molds  are  capable  of 
utilizing  pentoses  as  the  only  source  of  carbon,  while,  on  the  other  hand, 
other  organisms  are  quite  incapable  of  doing  so.  The  difference  in  food 
value  of  the  various  hexoses  are  well  known. 

Czapek  *  gives  the  following  values  for  Aspergillus  niger  in  weight  of 
yields : 


mg. 

d-fructose 523.7 

1-xylose 512.7 

d-galactose 489.3 


mg. 

d-glucose 477.1 

1-arabinose 350.0 

d-mannose  286.8 


1  CHALMONT,  G.  DE.    Pentosans  in  plants,  1.     (II)  Amer.  Chem.  Jour.,  16,  218-229, 

589-611,  1894. 
.    Die  Bildung  der  Pentosane  in  den  Pflanzen.    Ber.  d.  deut.  chem.  Ges.,  27, 

2722-2725,  1895. 
•TOLLENS,  B.     Ueber  die  Kohlehydrate  des  Maizes  und   der  Gerste  mit  besond. 

Beruecksichtigung  der  Pentosane.    Chem.  Zentr.,  69,  II,  967-968,  1898. 
1  WINDISH,  W.,  and  R.  HASSE.    Ueber  den  Pentosangehalt  der  Gerste  und  des  Maizes, 

inbesondere  ueber  das  Verhalten  der  Pentosane  bei  der  Keimung.    Chem. 

Zentr.,  72,  II,  1098-1099,  1901. 
*SPOEHB,  H.  A.    The  pentose  sugars  in  plant  metabolism.    The  Plant  World,  20, 

365-379, 1917. 
•CZAPEK,  F.    Biochemie  der  Pflanzen:   I.    Page  311,  1913.    Jena. 


THE  ORIGIN  AND  ROLE  OF  PENTOSE  SUGARS.  77 

The  high  nutritive  value  of  1-xylose  is  quite  evident,  while  on  the  other 
hand  it  is  well  known  that  the  yeasts  are  quite  incapable  of  utilizing  any 
of  the  pentose  sugars.  At  the  same  time  it  must  be  remembered  that  no 
one  of  the  sugars  is  universally  good  nutrient  material.  Little  is  known  of 
the  role  of  the  pentoses  in  the  metabolism  of  the  higher  plants.  The 
pentosans  in  the  walls  and  vessels  have  been  very  extensively  investigated, 
but  very  little  is  known  of  the  origin  and  physiological  role  of  these  sub- 
stances in  the  plant. 

In  the  mammalian  body  the  pentoses  have  been  found  to  be  about  iso- 
dynamic  in  food  value  with  the  fats,  but  are  not  protein  sparers  as  the 
hexoses  are.1  In  carnivorous  animals  as  high  as  50  to  60  per  cent  of  the 
amount  fed  has  been  observed  excreted  in  the  urine ;  in  omnivorous  animals 
the  percentage  is  less,  while  the  herbivorous  are  capable  of  utilizing  rela- 
tively large  quantities. 

That  the  pentose  sugars  are  consumed  at  a  very  appreciable  rate  in  the 
course  of  catabolism  of  the  cacti  was  shown  in  the  starvation  experiments 
previously  discussed.  It  was  also  demonstrated  that  when  the  cacti  are 
deprived  of  water  there  takes  place,  along  with  increase  in  the  amount  of 
polysaccharides,  a  very  decided  increase  in  pentose  sugars.  As  yet  we  have 
very  little  definite  knowledge  of  a  coordination  between  results  obtained  on 
the  rate  of  respiration  and  the  carbohydrate  economy  as  influenced  by  con- 
ditions such  as  water-balance  and  temperature.  No  simple  or  constant  rela- 
tion seems  to  exist  between  the  rate  of  carbon-dioxid  emission  and  the  supply 
of  carbohydrates.  It  is  most  probable  that  in  the  protein-carbohydrate 
complex  the  clavis  to  this  relationship  must  be  sought.  Furthermore,  the 
rate  of  respiration  can  not  be  constantly  or  definitely  associated  with  any 
one  group  of  sugars.  It  has  been  shown  that  the  hexose  monosaccharides, 
which  have  been  commonly  assumed  to  be  most  directly  involved  in  the 
respiratory  process,  are  at  times  present  in  only  exceedingly  small  amounts. 
This  is  particularly  the  case  under  conditions  of  low  water-content  and  of 
high  temperatures.  In  spite  of  the  fact  that  the  monosaccharide-content 
has  been  so  greatly  reduced,  the  rate  of  respiration  does  not  show  any  marked 
diminution.  It  would  seem,  therefore,  that  under  stress  the  plant  possesses 
the  power  of  utilizing  the  polysaccharides  and  aplastic  material.  An  indi- 
cation of  this  has  already  been  obtained  by  MacDougal,  Long,  and  Brown, 
in  the  study  already  referred  to,  on  cacti  which  had  been  starved  for  a  long 
time. 

The  formation  of  pentosans  is  intimately  associated  with  this  condition. 
As  has  been  stated,  the  simpler  sugars  decrease  in  amount  as  the  water- 
content  is  reduced,  and  vice  versa,  an  increase  in  water-supply  results  in  an 
increase  in  these  sugars.  Furthermore,  pentosan  formation  is  also  depen- 
dent upon  the  water-content  of  the  plant.  With  continued  low  water-supply 
the  pentosans  increase  decidedly. 

1  SCHEBOKITCH,  P.    Beitrag  zur  Bedeutung  der  Pentosen  als  Energte  quelle  im  tieri- 
schem  Organisms.    Biochem.  Zeitschr.,  55,  370-392,  1913. 


78 


THE  CARBOHYDRATE  ECONOMY  OF  CACTI. 


In  the  aldose  monosaccharides  the  first  carbon  atom  or  the  carbonyl 
group,  —  OH :  O,  is  the  most  reactive  and  is  largely  responsible  for  the 
great  reactivity  of  these  sugars.  In  the  disaccharides  and  polysaccharides 
found  in  these  plants  this  active  carbonyl  group  is  so  united  with  other 
groups  that  it  no  longer  forms  the  point  of  attack  in  chemical  reaction, 
These  sugars  are  therefore  first  affected  on  the  opposite  end  of  the  chain  of 
carbon  atoms,  at  the  —  CH2OH  group.1  Such  a  reaction  results  in  a 
primary  formation  of  glucuronic  acid,  CH1:  O(CHOH)4  •  OOOH. 

This  substance  has  been  found  as  a  product  of  glucose  metabolism  in 
mammals.  When  substances  such  as  chloral  or  camphor,  which  unite  with 
the  carbonyl  group  of  glucose,  are  fed,  glucuronic  acid,  conjugated  with 
these  substances,  is  found  in  the  urine.  It  has  been  established  now  that 
glucuronic  acid  is  also  present  in  the  cacti.  The  discovery  of  this  substance 
in  plants  is  especially  significant,  because  it  permits  the  formulation  of  a 
rational  theory  of  the  formation  of  pentoses  in  plants. 

A  very  general  property  of  acids  of  this  character  is  the  splitting-off  of 
CO2  from  the  carboxyl  group  when  solutions  thereof  are  exposed  to  the 
sunlight.  An  example  of  this  action  has  already  been  furnished  in  the 
case  of  malic  acid,  which  loses  two  molecules  of  carbon  dioxid  to  form  ethyl 
alcohol.1 

In  this  manner  glucuronic  acid  would  form  1-xylose : 


OH 


OH  OH 


OH 


OH 


COH- 


-COOH 


COH- 


-CH3OH 


H    OH   H    H  H    OH   H  +C0a 

Solkowski  and  Neuberg  *  have  reported  the  breaking-down  of  glucuronic 
acid  into  1-xylose  and  carbon  dioxid  by  means  of  bacteria. 

Further  support  of  this  interpretation  of  the  formation  of  pentoses  is 
obtained  from  a  consideration  of  the  structural  relations  of  the  various 
sugars  concerned.  If  the  pentoses  were  derived  from  a  direct  oxidation  of 
the  hexoses,  d-glueose  would  yield  d-arabinose  and  d-galactose  give  d-lyxose. 


OH 


OH      OH 


H 


OH       OH 


POTT 

i 

[         0 

H       I 

[          1 

1                                       C 

H       I 

I          1 

I 

D-glucose.                                                         D-arabinose. 

1  FISCHEB,  E.,  and  O.  PILOTT.    Reduction  der  Zuckersaeure.    Ber.  d.  deut.  chem.  Ges., 

24,  524,  1891, 

a  SPOEHB,  H.  A.     Biochem.  Zeitschr.,  57,  101,  1913. 
*  SOLKOWSKI,    E.,   and   C.   NEUBEEG.     Die   Verwandlung  von    d-glucuronsaeure   in 

1-xylose.    Chem.  Zentr.,  1902,  1,  1098. 


THE  ORIGIN  AND  ROLE   OF  PENTOSE  SUGARS. 
OH        H         H        OH  H         H         OH 


79 


COH- 


-CH,OH 


COH- 


H         OH       OH        H 
D-galactose. 


CH.OH 


OH       OH       H 
D-lyrose. 


It  is  a  striking  fact,  however,  that  in  nature  d-glucose  has  almost  always 
been  found  with  1-xylose,  and  that  d-galactose  is  usually  associated  with 
1-arabinose.  Also,  it  is  noteworthy  that  the  pentose  sugars  thus  found  are 
of  the  1-  and  not  of  the  d-  series.  These  facts  speak  strongly  in  favor  of  the 
theory  of  pentose  formation  here  presented.  A  study  of  the  photolysis  of 
the  hexonic  acids  which  is  now  in  progress  will  throw  much  light  on  these 
reactions. 

Finally,  a  brief  consideration  may  be  given  to  the  significance  of  such 
pentosan  formation  to  the  plant.  A  most  striking  property  of  the  pentosans 
is  their  mucilaginous  character.  They  have  the  power  of  swelling  and 
taking  up  an  enormous  amount  of  water.  This  property  is  not  exhibited  by 
the  hexose-polysaccharides  in  nearly  so  marked  a  degree.  A  notable  increase 
of  pentosans  in  the  cells  would  have  most  important  consequences.  The 
enormous  imbibition  capacity  of  the  pentosans  would  permit  the  absorption 
of  large  quantities  of  water  at  such  times  when  this  is  available.  In  other 
words,  the  presence  within  the  cell  of  a  hydrophile  colloid  such  as  a  pento- 
san makes  possible  the  absorption  of  large  quantities  of  water.  Such,  in 
fact,  is  the  nature  and  behavior  of  succulent  plants  as  the  cacti.  This  point 
is  of  special  interest  in  view  of  the  fact  that  the  hydrophile  colloids  in  the 
plants  are  themselves  products  of  arid  conditions,  and  opens  the  way  for  a 
new  conception  as  to  the  origin  of  the  morphological  character  of  succulent 
plants,  based  upon  physiological  principles. 


ITY  OF  CALIFORNIA 

Santa  Barbara  College  Library 
Santa  Barbara,  California 

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